Subpopulation Of Mesenchymal Stem Cells Research Articles

Enhancement of the function of mesenchymal stem cells by using a GMP-grade three-dimensional hypoxic large-scale production system

BackgroundEfficiently increasing the production of clinical-grade mesenchymal stem cells (MSCs) is crucial for clinical applications. Challenges with the current planar culture methods include scalability issues, labour intensity, concerns related to cell senescence, and heterogeneous responses. This study aimed to establish a large-scale production system for MSC generation. In addition, a comparative analysis of the biological differences between MSCs cultured under various conditions was conducted. Methods and materialsWe developed a GMP-grade three-dimensional hypoxic large-scale production (TDHLSP) system for MSCs using self-fabricated glass microcarriers and a multifunctional bioreactor. Different parameters, including cell viability, cell diameter, immunophenotype, morphology, karyotype, and tumourigenicity were assessed in MSCs cultured using different methods. Single-cell RNA sequencing (scRNA-seq) revealed pathways and genes associated with the enhanced functionality of MSCs cultured in three dimensions under hypoxic conditions (3D_Hypo MSCs). Moreover, CD142 knockdown in 3D_Hypo MSCs confirmed its in vitro functions. ResultsInoculating 2×108 MSCs into a 2.6 L bioreactor in the TDHLSP system resulted in a final scale of 4.6×109 3D_Hypo MSCs by day 10. The 3D_Hypo MSCs retained characteristics of the 2D MSCs, demonstrating their genomic stability and non-tumourigenicity. Interestingly, the subpopulations of 3D_Hypo MSCs exhibited a more uniform distribution and a closer relationship than those of 2D MSCs. The heterogeneity of MSCs was strongly correlated with 'cell cycle' and 'stroma/mesenchyme', with 3D_Hypo MSCs expressing higher levels of activated stroma genes. Compared to 2D MSCs, 3D_Hypo MSCs demonstrated enhanced capabilities in blood vessel formation, TGF-β1 secretion, and inhibition of BV2 proliferation, with maintenance of Senescence-Associated β-Galactosidase (SA-β-gal) negativity. However, the enhanced functions of 3D_Hypo MSCs decreased upon the downregulation of CD142 expression. ConclusionThe TDHLSP system led to a high overall production of MSCs and promoted uniform distribution of MSC clusters. This cultivation method also enhanced key cellular properties, such as angiogenesis, immunosuppression, and anti-aging. These functionally improved and uniform MSC subpopulations provide a solid basis for the clinical application of stem cell therapies.

Vascular architecture regulates mesenchymal stromal cell heterogeneity via P53-PDGF signaling in the mouse incisor.

Mesenchymal stem cells (MSCs) reside in niches to maintain tissue homeostasis and contribute to repair and regeneration. Although the physiological functions of blood and lymphatic vasculature are well studied, their regulation of MSCs as niche components remains largely unknown. Using adult mouse incisors as a model, we uncover the role of Trp53 in regulating vascular composition through THBS2 to maintain mesenchymal tissue homeostasis. Loss of Trp53 in GLI1+ progeny increases arteries and decreases other vessel types. Platelet-derived growth factors from arteries deposit in the MSC region and interact with PDGFRA and PDGFRB. Significantly, PDGFRA+ and PDGFRB+ cells differentially contribute to defined cell lineages in the adult mouse incisor. Collectively, our results highlight Trp53's importance in regulating the vascular niche for MSCs. They also shed light on how different arterial cells provide unique cues to regulate MSC subpopulations and maintain their heterogeneity. Furthermore, they provide mechanistic insight into MSC-vasculature crosstalk.

Mesenchymal stem cells induce an immunosuppressive microenvironment in pituitary tumors.

The tumor microenvironment (TME) includes diverse cellular components such as mesenchymal stem cells (MSC) and immune cells among others. MSC have been isolated from different tumors and they favor tumor cell growth, however, their role in pituitary tumors (PT) remains unknown. Herein we report the presence of MSCs in 2 ACTH-secreting PT causing Cushing disease (MCU), 2 nonfunctioning adenomas of gonadotrope differentiation (MNF) and 2 non tumoral pituitary glands (MS). We have analyzed their transcriptomic profiles by RNAseq and compared MSC in terms of their immunosuppressive effects against lymphoid T cell and macrophage populations by means of co-cultures and flow cytometry. Our transcriptomic analysis revealed molecular differences between MSC derived from non-tumoral pituitaries and MSC derived from PT. Two distinct subpopulations of MSC, one displaying immunosuppressive properties and the other with increased pro-proliferative capabilities, regardless of their origin. MSC derived from ACTH- and nonfunctioning PT, but not those derived from non-tumoral glands significantly inhibited the proliferation of activated T cells, favored the generation of Tregs and promote M2 macrophage polarization. Such immunosuppressive effects were correlated with an upregulation of programmed death ligand 1 and intracellular expression of macrophage colony stimulating factor (M-CSF) and IL-10. Importantly, MSC derived from ACTH-PT showed a higher immunosuppressive potential than MSC isolated from nonfunctioning tumors. This study demonstrates the presence of at least two MSC subpopulations in the pituitary gland and suggests that immunosuppressive effects of MSC may have important implications in PT growth.

Mgp High-Expressing MSCs Orchestrate the Osteoimmune Microenvironment of Collagen/Nanohydroxyapatite-Mediated Bone Regeneration.

Activating autologous stem cells after the implantation of biomaterials is an important process to initiate bone regeneration. Although several studies have demonstrated the mechanism of biomaterial-mediated bone regeneration, a comprehensive single-cell level transcriptomic map revealing the influence of biomaterials on regulating the temporal and spatial expression patterns of mesenchymal stem cells (MSCs) is still lacking. Herein, the osteoimmune microenvironment is depicted around the classical collagen/nanohydroxyapatite-based bone repair materials via combining analysis of single-cell RNA sequencing and spatial transcriptomics. A group of functional MSCs with high expression of matrix Gla protein (Mgp) is identified, which may serve as a pioneer subpopulation involved in bone repair. Remarkably, these Mgp high-expressing MSCs (MgphiMSCs) exhibit efficient osteogenic differentiation potential and orchestrate the osteoimmune microenvironment around implanted biomaterials, rewiring the polarization and osteoclastic differentiation of macrophages through the Mdk/Lrp1 ligand-receptor pair. The inhibition of Mdk/Lrp1 activates the pro-inflammatory programs of macrophages and osteoclastogenesis. Meanwhile, multiple immune-cell subsets also exhibit close crosstalk between MgphiMSCs via the secreted phosphoprotein 1 (SPP1) signaling pathway. These cellular profiles and interactions characterized in this study can broaden the understanding of the functional MSC subpopulations at the early stage of biomaterial-mediated bone regeneration and provide the basis for materials-designed strategies that target osteoimmune modulation.

Intranasal delivery of small extracellular vesicles from specific subpopulation of mesenchymal stem cells mitigates traumatic spinal cord injury

Vascular injury following spinal cord injury (SCI) can significantly exacerbate secondary SCI and result in neurological dysfunction. Strategies targeting angiogenesis have demonstrated potential in enhancing functional recovery post-SCI. In the context of angiogenesis, the CD146+ and CD271+ subpopulations of mesenchymal stem cells (MSCs) have been recognized for their angiogenic capabilities in tissue repair. Small extracellular vesicles (sEVs) derived from MSCs are nanoscale vesicles containing rich bioactive components that play a crucial role in tissue regeneration. However, the precise role of sEVs derived from CD146+CD271+ UCMSCs (CD146+CD271+ UCMSC-sEVs) in SCI remain unclear. In this study, CD146+CD271+ UCMSC-sEVs were non-invasively administered via intranasal delivery, demonstrating a significant capacity to stimulate angiogenesis and improve functional recovery in mice following SCI. Furthermore, in vitro assessments revealed the effective enhancement of migration and tube formation capabilities of the murine brain microvascular endothelial cell line (bEnd.3) by CD146+CD271+UCMSC-sEVs. MicroRNA array analysis confirmed significant enrichment of multiple microRNAs within CD146+CD271+ UCMSC-sEVs. Subsequent in vivo and in vitro experiments demonstrated that CD146+CD271+ UCMSC-sEVs promote enhanced angiogenesis and improved functional recovery mediated by miR-27a-3p. Further mechanistic studies revealed that miR-27a-3p sourced from CD146+CD271+ UCMSC-sEVs enhances migration and tube formation of bEnd.3 cells in vitro by suppressing the expression of Delta Like Canonical Notch Ligand 4 (DLL4), thereby promoting angiogenesis in vivo. Collectively, our results demonstrate that a crucial role of CD146+CD271+ UCMSC-sEVs in inhibiting DLL4 through the transfer of miR-27a-3p, which leads to the promotion of angiogenesis and improved functional recovery after SCI.

Size-Based Microfluidic-Enriched Mesenchymal Stem Cell Subpopulations Enhance Articular Cartilage Repair

Background: The functional heterogeneity of culture-expanded mesenchymal stem cells (MSCs) has hindered the clinical application of MSCs. Previous studies have shown that MSC subpopulations with superior chondrogenic capacity can be isolated using a spiral microfluidic device based on the principle of inertial cell focusing. Hypothesis: The delivery of microfluidic-enriched chondrogenic MSCs that are consistent in size and function will overcome the challenge of the functional heterogeneity of expanded MSCs and will significantly improve MSC-based cartilage repair. Study Design: Controlled laboratory study. Methods: A next-generation, fully automated multidimensional double spiral microfluidic device was designed to provide more refined and efficient isolation of MSC subpopulations based on size. Analysis of in vitro chondrogenic potential and RNA sequencing was performed on size-sorted MSC subpopulations. In vivo cartilage repair efficacy was demonstrated in an osteochondral injury model in 12-week-old rats. Defects were implanted with MSC subpopulations (n = 6 per group) and compared with those implanted with unsegregated MSCs (n = 6). Osteochondral repair was assessed at 6 and 12 weeks after surgery by histological, micro–computed tomography, and mechanical analysis. Results: A chondrogenic MSC subpopulation was efficiently isolated using the multidimensional double spiral device. RNA sequencing revealed distinct transcriptomic profiles and identified differential gene expression between subpopulations. The delivery of a chondrogenic MSC subpopulation resulted in improved cartilage repair, as indicated by histological scoring, the compression modulus, and micro–computed tomography of the subchondral bone. Conclusion: We have established a rapid, label-free, and reliable microfluidic protocol for more efficient size-based enrichment of a chondrogenic MSC subpopulation. Our proof-of-concept in vivo study demonstrates the enhanced cartilage repair efficacy of these enriched chondrogenic MSCs. Clinical Relevance: The delivery of microfluidic-enriched chondrogenic MSCs that are consistent in size and function can overcome the challenge of the functional heterogeneity of expanded MSCs, resulting in significant improvement in MSC-based cartilage repair. The availability of such rapid, label-free enriched chondrogenic MSCs can enable better cell therapy products for cartilage repair with improved treatment outcomes.

Sorting Technology for Mesenchymal Stem Cells from a Single Tissue Source.

Mesenchymal stem cells (MSCs) are adult stem cells that can be obtained, enriched and proliferated in vitro. They owned enormous potential in fields like regenerative medicine, tissue engineering and immunomodulation. However, though isolated from the same origin, MSCs are still essentially heterogeneous cell populations with different phenotypes and functions. This heterogeneity of MSCs significantly affects their therapeutic efficacy and brings obstacles to scientific research. Thus, reliable sorting technology which can isolate or purify MSC subpopulations with various potential and differentiation pathways is urgently needed. This review summarized principles, application status and clinical implications for these sorting methods, aiming at improving the understanding of MSC heterogeneity as well as providing fresh perspectives for subsequent clinical applications.

CD_99 G1 neutrophils modulate osteogenic differentiation of mesenchymal stem cells in the pathological process of ankylosing spondylitis

ObjectivesThis study aimed to identify the types and heterogeneity of cells within the spinal enthesis and investigate the underlying mechanisms of osteogenesis.MethodsSingle-cell RNA sequencing was used to identify cell populations...

Single-cell sequencing analysis reveals the relationship between tumor microenvironment cells and oxidative stress in breast cancer bone metastases.

Bone metastasis (BM) is one of the main manifestations of advanced breast cancer (BC), causing complications such as pathological fractures, which seriously affects the quality of life of patients and even leads to death. In our study, a global single-cell landscape of the tumor microenvironment was constructed using single cell RNA sequencing data from BM. BC cells were found to be reduced in the BM, while mesenchymal stem cells (MSCs), Fibroblasts and other cells were significantly more abundant in the BM. The subpopulations of these cells were further identified, and the pathways, developmental trajectories and transcriptional regulation of different subpopulations were discussed. The results suggest that with the development of BM, BC cells were vulnerable to oxidative damage, showing a high level of oxidative stress, which played a key role in cell apoptosis. Fibroblasts were obviously involved in the biological processes (BPs) related to ossification and bone remodeling, and play an important role in tumor cell inoculation to bone marrow and growth. MSC subpopulations were significantly enriched in a number of BPs associated with bone growth and development and oxidative stress and may serve as key components of BC cells homing and adhesion to the ecological niche of BM. In conclusion, our research results describe the appearance of tumor microenvironment cell subpopulations in breast cancer patients, reveal the important role of some cells in the balance of BM bone remodeling and the imbalance of BM development, and provide potential therapeutic targets for BM.

Crosstalk between Mesenchymal Stem Cells and Cancer Stem Cells Reveals a Novel Stemness-Related Signature to Predict Prognosis and Immunotherapy Responses for Bladder Cancer Patients

Mesenchymal stem cells (MSCs) and cancer stem cells (CSCs) maintain bladder cancer (BCa) stemness and facilitate the progression, metastasis, drug resistance, and prognosis. Therefore, we aimed to decipher the communication networks, develop a stemness-related signature (Stem. Sig.), and identify a potential therapeutic target. BCa single-cell RNA-seq datasets (GSE130001 and GSE146137) were used to identify MSCs and CSCs. Pseudotime analysis was performed by Monocle. Stem. Sig. was developed by analyzing the communication network and gene regulatory network (GRN) that were decoded by NicheNet and SCENIC, respectively. The molecular features of the Stem. Sig. were evaluated in TCGA-BLCA and two PD-(L)1 treated datasets (IMvigor210 and Rose2021UC). A prognostic model was constructed based on a 101 machine-learning framework. Functional assays were performed to evaluate the stem traits of the hub gene. Three subpopulations of MSCs and CSCs were first identified. Based on the communication network, the activated regulons were found by GRN and regarded as the Stem. Sig. Following unsupervised clustering, two molecular subclusters were identified and demonstrated distinct cancer stemness, prognosis, immunological TME, and response to immunotherapy. Two PD-(L)1 treated cohorts further validated the performance of Stem. Sig. in prognosis and immunotherapeutic response prediction. A prognostic model was then developed, and a high-risk score indicated a poor prognosis. Finally, the hub gene SLC2A3 was found exclusively upregulated in extracellular matrix-related CSCs, predicting prognosis, and shaping an immunosuppressive tumor microenvironment. Functional assays uncovered the stem traits of SLC2A3 in BCa by tumorsphere formation and western blotting. The Stem. Sig. derived from MSCs and CSCs can predict prognosis and response to immunotherapy for BCa. Besides, SLC2A3 may serve as a promising stemness target facilitating cancer effective management.

How Mechanical and Physicochemical Material Characteristics Influence Adipose-Derived Stem Cell Fate

Adipose-derived stem cells (ASCs) are a subpopulation of mesenchymal stem cells. Compared to bone marrow-derived stem cells, they can be harvested with minimal invasiveness. ASCs can be easily expanded and were shown to be able to differentiate into several clinically relevant cell types. Therefore, this cell type represents a promising component in various tissue engineering and medical approaches (e.g., cell therapy). In vivo cells are surrounded by the extracellular matrix (ECM) that provides a wide range of tissue-specific physical and chemical cues, such as stiffness, topography, and chemical composition. Cells can sense the characteristics of their ECM and respond to them in a specific cellular behavior (e.g., proliferation or differentiation). Thus, in vitro biomaterial properties represent an important tool to control ASCs behavior. In this review, we give an overview of the current research in the mechanosensing of ASCs and current studies investigating the impact of material stiffens, topography, and chemical modification on ASC behavior. Additionally, we outline the use of natural ECM as a biomaterial and its interaction with ASCs regarding cellular behavior.

Immunohistochemical analysis of selected stem cell markers including stage-specific mouse embryonic antigen 1 (SSEA-1) in porcine and bovine endometrium throughout the oestrous cycle

There has been a lack of research regarding endometrial stem cells in farm animals, and particularly, regarding epithelial stem/progenitor cells. We aimed to identify potential stem cell subpopulations in porcine and bovine endometrium by measuring the expression of selected stem cell markers (OCT3/4, CD44, SSEA-1, SOX10, CD73, and CD117) during the follicular and luteal phases of the oestrous cycle. We studied 28 endometrial tissue samples collected from 14 healthy, cycling pigs and cows. The endometrial mucosa of each sample was divided into basal, middle, and luminal portions, and the immunohistochemical staining intensity and percentages of cells that were marker-positive were recorded for each portion of the endometrium. Expression of OCT-3/4 was identified in the glands and stroma, and that of SOX10 and CD117 was identified in stroma of both porcine and bovine endometrium. In porcine endometrium, CD44 was only expressed in the glands, whereas SSEA-1 was expressed in the glands and stroma. In bovine endometrium, CD73 was only expressed in the glands. Differences in the expression of most of these markers were found between phases of the oestrous cycle and/or portions of the endometrial mucosa. Our data indicate the existence of both epithelial and mesenchymal stem cell subpopulations in the endometrium of pigs and cows during their oestrous cycles. The expression pattern of the stem cell marker SSEA-1 in porcine endometrium suggests a possible alternative location of the epithelial stem/progenitor cell population in the middle portion of the endometrial glands.

Intravenous administration of human Muse cells recovers blood flow in a mouse model of hindlimb ischemia.

Cell-based therapies hold great promise for the treatment of peripheral arterial disease (PAD), especially in patients presenting with severe limb ischemia, although the optimal strategy remains to be explored. In this study, we evaluated the therapeutic effect of intravenous administration of human Muse cells, a unique subpopulation of mesenchymal stem cells (MSC), using a mouse model of hindlimb ischemia (HLI) without an immunosuppressant. Compared with the phosphate buffered saline (PBS) or non-Muse MSC groups, the Muse group showed significantly higher laser doppler blood flow in the ischemic limb at days 7 and 14 after HLI. Increased microvascular density [percent area of CD31(+) cells] and reduced interstitial fibrosis in the ischemic limb muscle were also observed in the Muse group. mCherry-expressing Muse cells were found in the ischemic border zone and expressed CD31 but did not in the non-ischemic limb. Muse cells produced higher amounts of vascular endothelial growth factor (VEGF) than non-Muse cells under normoxic and hypoxic conditions in vitro. In the ischemic muscle, tissue VEGF concentration and angiogenesis-related genes such as Vegfa, Angpt1, Pdgfb, and Igf1 were significantly higher in the Muse group than in the other two groups. In addition, the proportion of M2 macrophages to total macrophages and the ratio of anti-inflammatory-related genes such as IL-10, Arg1, and CD206 per iNOS were significantly higher in the Muse group than in the other two groups. In summary, Muse cells exert pleiotropic effects in a mouse model of HLI, and therefore may provide a novel therapeutic approach for the treatment of PAD patients with severe limb ischemia.

Abstract 10174: Intravenous Muse Cell Transplantation Enhances Angiogenesis and Reduces Muscle Fibrosis in a Mouse Hindlimb Ischemia Model

Introduction: Multilineage-differentiating stress enduring (Muse) cells, a unique subpopulation of mesenchymal stem cells (MSCs), have been proposed as a promising cell source for regenerative medicine. However, the therapeutic potential of the Muse cells in ischemic limb disease remains unclear. In this study, we aimed to investigate therapeutic potential of Muse cell transplantation for the treatment of severe limb ischemia. Methods and Results: Muse cells isolated from human MSCs, non-Muse MSCs, or mouse bone marrow-derived mononuclear cells (BM-MNCs) were administered intravenously or intramuscularly to a mouse model of hindlimb ischemia (HLI). When the same number of cells were administered, blood flow recovery of the ischemic limb was significantly enhanced in the mice administered with Muse cells over 2 weeks compared to the others. Intravenous Muse cell administration resulted in comparable blood flow recovery to intramuscular Muse cell administration. Immunohistochemistry analysis revealed that intravenous Muse cell administration increased microvascular density and ameliorated interstitial fibrosis in the ischemic limb. We detected mCherry-labeled Muse cells expressing CD31 and human genomic DNA in the ischemic limb 1 week after the intravenous Muse cell administration. Muse cells produced higher VEGF compared to non-Muse MSCs, and Muse cell administration increased tissue VEGF level and expression of angiogenesis-related genes in the ischemic limb. Moreover, we found that Muse cell administration increased M2 macrophage and gene expression ratio of anti-inflammatory genes to proinflammatory gene, iNOS, in the ischemic limb. Lastly, we administered Muse cell intravenously at day 1 and 3, or high-dose BM-MNCs intramuscularly at day1 after the HLI surgery and found that repetitive Muse cell administration resulted in significantly enhanced blood flow recovery in the ischemic limb compared to high-dose BM-MNCs administration. Conclusions: Using an animal model, we demonstrated that intravenous Muse cell transplantation exerts robust therapeutic effects on limb ischemia injury. Therefore, Muse cell-based therapy may provide a novel therapeutic approach for patients with severe limb ischemia.

Functional Heterogeneity of Bone Marrow Mesenchymal Stem Cell Subpopulations in Physiology and Pathology.

Bone marrow mesenchymal stem cells (BMSCs) are multi-potent cell populations and are capable of maintaining bone and body homeostasis. The stemness and potential therapeutic effect of BMSCs have been explored extensively in recent years. However, diverse cell surface antigens and complex gene expression of BMSCs have indicated that BMSCs represent heterogeneous populations, and the natural characteristics of BMSCs make it difficult to identify the specific subpopulations in pathological processes which are often obscured by bulk analysis of the total BMSCs. Meanwhile, the therapeutic effect of total BMSCs is often less effective partly due to their heterogeneity. Therefore, understanding the functional heterogeneity of the BMSC subpopulations under different physiological and pathological conditions could have major ramifications for global health. Here, we summarize the recent progress of functional heterogeneity of BMSC subpopulations in physiology and pathology. Targeting tissue-resident single BMSC subpopulation offers a potentially innovative therapeutic strategy and improves BMSC effectiveness in clinical application.

An optimized method for obtaining clinical-grade specific cell subpopulations from human umbilical cord-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are heterogeneous populations with broad application prospects in cell therapy, and using specific subpopulations of MSCs can enhance their particular capability under certain conditions and achieve better therapeutic effects. However, no studies have reported how to obtain high‐quality specific MSC subpopulations in vitro culture. Here, for the first time, we established a general operation process for obtaining high‐quality clinical‐grade cell subpopulations from human umbilical cord MSCs (hUC‐MSCs) based on particular markers. We used the MSC‐CD106+ subpopulations, whose biological function has been well documented, as an example to explore and optimize the crucial links of primary preparation, pre‐treatment, antibody incubation, flow sorting, quality and function test. After comprehensively evaluating the quality and function of the acquired MSC‐CD106+ subpopulations, including in vitro cell viability, apoptosis, proliferation, marker stability, adhesion ability, migration ability, tubule formation ability, immunomodulatory function and in vivo wound healing ability and proangiogenic activity, we defined an important pre‐treatment scheme which might effectively improve the therapeutic efficiency of MSC‐CD106+ subpopulations in two critical clinical application scenarios—direct injection after cell sorting and post‐culture injection into bodies. Based on the above, we tried to establish a general five‐step operation procedure for acquiring high‐quality clinical‐grade MSC subpopulations based on specific markers, which cannot only improve their enrichment efficiency and the reliability of preclinical studies, but also provide valuable methodological guidance for the rapid clinical transformation of specific MSC subpopulations.

Single-cell RNA sequencing analysis of human bone-marrow-derived mesenchymal stem cells and functional subpopulation identification.

Mesenchymal stem cells (MSCs) are a common kind of multipotent cell in vivo, but their heterogeneity limits their further applications. To identify MSC subpopulations and clarify their relationships, we performed cell mapping of bone-marrow-derived MSCs through single-cell RNA (scRNA) sequencing. In our study, three main subpopulations, namely, the stemness subpopulation, functional subpopulation, and proliferative subpopulation, were identified using marker genes and further bioinformatic analyses. Developmental trajectory analysis showed that the stemness subpopulation was the root and then became either the functional subpopulation or the proliferative subpopulation. The functional subpopulation showed stronger immunoregulatory and osteogenic differentiation abilities but lower proliferation and adipogenic differentiation. MSCs at different passages or isolated from different donors exhibited distinct cell mapping profiles, which accounted for their corresponding different functions. This study provides new insight into the biological features and clinical use of MSCs at the single-cell level, which may contribute to expanding their application in the clinic.

Single-cell Transcriptomic Analysis Reveals the Cellular Heterogeneity of Mesenchymal Stem Cells

Ex vivo-expanded mesenchymal stem cells (MSCs) have been demonstrated to be a heterogeneous mixture of cells exhibiting varying proliferative, multipotential, and immunomodulatory capacities. However, the exact characteristics of MSCs remain largely unknown. By single-cell RNA sequencing of 61,296 MSCs derived from bone marrow and Wharton’s jelly, we revealed five distinct subpopulations. The developmental trajectory of these five MSC subpopulations was mapped, revealing a differentiation path from stem-like active proliferative cells (APCs) to multipotent progenitor cells, followed by branching into two paths: 1) unipotent preadipocytes or 2) bipotent prechondro-osteoblasts that were subsequently differentiated into unipotent prechondrocytes. The stem-like APCs, expressing the perivascular mesodermal progenitor markers CSPG4/MCAM/NES, uniquely exhibited strong proliferation and stemness signatures. Remarkably, the prechondrocyte subpopulation specifically expressed immunomodulatory genes and was able to suppress activated CD3+ T cell proliferation in vitro, supporting the role of this population in immunoregulation. In summary, our analysis mapped the heterogeneous subpopulations of MSCs and identified two subpopulations with potential functions in self-renewal and immunoregulation. Our findings advance the definition of MSCs by identifying the specific functions of their heterogeneous cellular composition, allowing for more specific and effective MSC application through the purification of their functional subpopulations.

WNT/beta-catenin signalling interrupts a senescence-induction cascade in human mesenchymal stem cells that restricts their expansion

Senescence, the irreversible cell cycle arrest of damaged cells, is accompanied by a deleterious pro-inflammatory senescence-associated secretory phenotype (SASP). Senescence and the SASP are major factors in aging, cancer, and degenerative diseases, and interfere with the expansion of adult cells in vitro, yet little is known about how to counteract their induction and deleterious effects. Paracrine signals are increasingly recognized as important senescence triggers and understanding their regulation and mode of action may provide novel opportunities to reduce senescence-induced inflammation and improve cell-based therapies. Here, we show that the signalling protein WNT3A counteracts the induction of paracrine senescence in cultured human adult mesenchymal stem cells (MSCs). We find that entry into senescence in a small subpopulation of MSCs triggers a secretome that causes a feed-forward signalling cascade that with increasing speed induces healthy cells into senescence. WNT signals interrupt this cascade by repressing cytokines that mediate this induction of senescence. Inhibition of those mediators by interference with NF-κB or interleukin 6 signalling reduced paracrine senescence in absence of WNT3A and promoted the expansion of MSCs. Our work reveals how WNT signals can antagonize senescence and has relevance not only for expansion of adult cells but can also provide new insights into senescence-associated inflammatory and degenerative diseases.

P8 - Polycaprolactone/Nano-Hydroxyapatite Nanofibrous Scaffold as a Cell Homing-Based Therapy

This study investigated the behavior of human dental pulp cells (HDPCs) seeded on the surface of polycaprolactone/nano-hydroxyapatite nanofibrous scaffolds, aiming a dentin tissue engineering-based vital pulp therapy (VPT). Polycaprolactone (PCL)-based solutions (10% w/v in 8:2 chloroform and dimethylformamide) containing nano-hydroxyapatite (nHA) particles (0.5; 1.0; or 2.0% w/v) were electrospun into nanofibrous scaffolds. PCL scaffolds without nHA were used as control. The scaffolds were blindly characterized for morphology and composition (SEM/EDS; n=12), solubility (n=6), the release of calcium/phosphate (n=6), and change of medium pH (n=6). A culture of HDPCs was obtained from sound third molars and characterized for the presence of a subpopulation of mesenchymal stem cells. Then, HDPCs were seeded on the surface of the scaffolds and evaluated for cell viability (alamarBlue; n=8), cell proliferation (Live/Dead assay; n=4), adhesion and spreading (F-actin; n=4), total protein content (TP; Lowry method; n=8), alkaline phosphatase activity (ALP; thymolphthalein assay; n=8), expression of odontogenic genes (RT-qPCR; DSPP, DMP1, COL1A1, and ALPL; n=6), and formation of a mineralized matrix (Alizarin Red staining; n=8). All experiments were repeated at least twice. Sample sizes were calculated using G-Power. Data were analyzed with confidence intervals and one- or two-way ANOVA followed by Tukey, Sidak, or Games-Howell post-hocs (?=5%). All formulations generated randomly arranged fibers ranging from 600 to 900 nm in diameter. Higher nHA concentrations roughened the surface of the nanofibers, while PCL+2%nHA increased the interfibrillar spaces. PCL+1%nHA and PCL+2%nHA significantly released calcium/phosphate over a period of 28 days in comparison with PCL+0.5%nHA. Nonetheless, the medium pH was maintained below 8.0. HDPCs viability and proliferation were not affected by the incorporation of nHA, while cell adhesion/spreading was stimulated, especially for PCL+2%nHA. Higher total protein content and increased ALP activity were seen in the presence of nHA. PCL+1%nHA and PCL+2%nHA upregulated the expression of DSPP and DMP1 in 14 days; and COL1A1, ALPL, and DMP1 in 21 days. The formation of a mineralized matrix after 21 days was concentration-dependent, and it was about 9× higher for PCL+2%nHA formulation compared to control.