Subpopulation Of Mesenchymal Stromal Cells Research Articles

Heterogeneity in Dental Tissue–Derived MSCs Revealed by Single-Cell RNA-seq

Mesenchymal stromal cells (MSCs) are multipotent, progenitor cells that reside in tissues across the human body, including the periodontal ligament (PDL) and gingiva. They are a promising therapeutic tool for various degenerative and inflammatory diseases. However, different heterogeneity levels caused by tissue-to-tissue and donor-to-donor variability, and even intercellular differences within a given MSCs population, restrict their therapeutic potential. There are considerable efforts to decipher these heterogeneity levels using different “omics” approaches, including single-cell transcriptomics. Previous studies applied this approach to compare MSCs isolated from various tissues of different individuals, but distinguishing between donor-to-donor and tissue-to-tissue variability is still challenging. In this study, MSCs were isolated from the PDL and gingiva of 5 periodontally healthy individuals and cultured in vitro. A total of 3,844 transcriptomes were generated using single-cell mRNA sequencing. Clustering across the 2 different tissues per donor identified PDL- and gingiva-specific and tissue-spanning MSCs subpopulations with unique upregulated gene sets. Gene/pathway enrichment and protein-protein interaction (PPI) network analysis revealed differences restricted to several cellular processes between tissue-specific subpopulations, indicating a limited tissue-of-origin variability in MSCs. Gene expression, pathway enrichment, and PPI network analysis across all donors’ PDL- or gingiva-specific subpopulations showed significant but limited donor-to-donor differences. In conclusion, this study demonstrates tissue- and donor-specific variabilities in the transcriptome level of PDL- and gingiva-derived MSCs, which seem restricted to specific cellular processes. Identifying tissue-specific and tissue-spanning subpopulations highlights the intercellular differences in dental tissue–derived MSCs. It could be reasonable to control MSCs at a single-cell level to ensure their properties before transplantation.

The influence of fetal bovine serum concentration on stemness and neuronal differentiation markers in stem cells from human exfoliated deciduous teeth

Dental Stem Cells (DSCs) from discarded teeth are a non-invasive and ethically favorable source with the potential for neurogenesis due to their ectodermal origin. Stem cells from human exfoliated deciduous teeth (SHED) are particularly promising due to their high differentiation potential and relative immaturity compared to other Mesenchymal Stromal Cells (MSCs). Markers like CD56 and CD271 are critical in identifying MSC subpopulations for therapeutic applications because of their roles in neurodevelopment and maintaining stemness. This study investigates how fetal bovine serum (FBS) concentrations affect the expression of CD56 and CD271 in SHED, influencing their stemness and neuronal differentiation potential. SHEDs were isolated from various donors, cultured, and characterized for MSC traits using markers such as CD14, CD19, CD29, CD34, CD45, CD73, CD90, CD105, CD56, and CD271. Culturing SHED in different FBS conditions (standard 15 %, reduced 1 % and 5 %, and FBS-free) showed that lower FBS concentrations increase CD271 and CD56 expression while maintaining the standard MSC immunophenotype. Importantly, the enhanced expression of these markers can be induced even after SHEDs have been expanded in standard FBS concentrations. These findings suggest that FBS concentration can optimize SHED culture conditions, enhancing their suitability for regenerative medicine and tissue engineering applications.

Single-cell RNA analysis of chromodomain-encoding genes in mesenchymal stromal cells of the mouse dental pulp.

The chromodomain helicase DNA-binding (CHD) and chromobox (CBX) families of proteins play crucial roles in cell fate decisions, differentiation, and cell proliferation in a broad variety of tissues and cell types. CHD proteins are ATP-dependent epigenetic enzymes actively engaged in transcriptional regulation, DNA replication, and DNA damage repair, whereas CBX proteins are transcriptional repressors mainly involved in the formation of heterochromatin. The pleiotropic effects of CHD and CBX proteins are largely dependent on their versatility to interact with other key components of the epigenetic and transcriptional machinery. Although the function and regulatory modes of CHD and CBX factors are well established in many cell types, little is known about their roles during osteogenic differentiation. A single-cell RNA-sequencing analysis of the mouse incisor dental pulp revealed distinct spatiotemporal expression patterns of CHD- and CBX-encoding genes within different clusters of mesenchymal stromal cells (MSCs) representing various stages of osteogenic differentiation. Additionally, genes encoding interaction partners of CHD and CBX proteins, such as subunits of the trithorax-COMPASS and polycomb chromatin remodeling complexes, exhibited differential co-expression behaviors within MSC subpopulations. Thus, CHD- and CBX-encoding genes show partially overlapping but distinct expression patterns in MSCs, suggesting their differential roles in osteogenic cell fate decisions.

MAPPING THE SPATIAL AND TEMPORAL HETEROGENEITY OF MESENCHYMAL STROMAL CELLS WHEN USED AS A CELL THERAPY IN OSTEOCHONDRAL REPAIR

Despite osteoarthritis (OA) representing a large burden for healthcare systems, there remains no effective intervention capable of regenerating the damaged cartilage in OA. Mesenchymal stromal cells (MSCs) are adult-derived, multipotent cells which are a candidate for musculoskeletal cell therapy. However, their precise mechanism of action remains poorly understood.The effects of an intra-articular injection of human bone-marrow derived MSCs into a knee osteochondral injury model were investigated in C57Bl/6 mice. The cell therapy was retrieved at different time points and single cell RNA sequencing was performed to elucidate the transcriptomic changes relevant to driving tissue repair. Mass cytometry was also used to study changes in the mouse immune cell populations during repair.Histological assessment reveals that MSC treatment is associated with improved tissue repair in C57Bl/6 mice. Single cell analysis of retrieved human MSCs showed spatial and temporal transcriptional heterogeneity between the repair tissue (in the epiphysis) and synovial tissue. A transcriptomic map has emerged of some of the distinct genes and pathways enriched in human MSCs isolated from different tissues following osteochondral injury. Several MSC subpopulations have been identified, including proliferative and reparative subpopulations at both 7 days and 28 days after injury. Supported by the mass cytometry results, the immunomodulatory role of MSCs was further emphasised, as MSC therapy was associated with the induction of increased numbers of regulatory T cells correlating with enhanced repair in the mouse knee.The transcriptomes of a retrieved MSC therapy were studied for the first time. An important barrier to the translation of MSC therapies is a lack of understanding of their heterogeneity, and the consequent lack of precision in its use. MSC subpopulations with different functional roles may be implicated in the different phases of tissue repair and this work offers further insights into repair process.

Associating growth factor secretions and transcriptomes of single cells in nanovials using SEC-seq.

Cells secrete numerous bioactive molecules that are essential for the function of healthy organisms. However, scalable methods are needed to link individual cell secretions to their transcriptional state over time. Here, by developing and using secretion-encoded single-cell sequencing (SEC-seq), which exploits hydrogel particles with subnanolitre cavities (nanovials) to capture individual cells and their secretions, we simultaneously measured the secretion of vascular endothelial growth factor A (VEGF-A) and the transcriptome for thousands of individual mesenchymal stromal cells. Our data indicate that VEGF-A secretion is heterogeneous across the cell population and is poorly correlated with the VEGFA transcript level. The highest VEGF-A secretion occurs in a subpopulation of mesenchymal stromal cells characterized by a unique gene expression signature comprising a surface marker, interleukin-13 receptor subunit alpha 2 (IL13RA2), which allowed the enrichment of this subpopulation. SEC-seq enables the identification of gene signatures linked to specific secretory states, facilitating mechanistic studies, the isolation of secretory subpopulations and the development of means to modulate cellular secretion.

Improving vasculoprotective effects of MSCs in coronary microvessels - benefits of 3D culture, sub-populations and heparin.

Opening occluded coronary arteries in patients with myocardial infarction (MI) damages the delicate coronary microvessels through a process called myocardial ischaemia-reperfusion injury. Although mesenchymal stromal cells (MSCs) have the potential to limit this injury, clinical success remains limited. This may be due to (i) poor MSC homing to the heart (ii) infused MSCs, even if derived from the same site, being a heterogeneous population with varying therapeutic efficacy and (iii) conventional 2D culture of MSCs decreasing their homing and beneficial properties. This study investigated whether 3D culture of two distinctly different bone marrow (BM)-derived MSC sub-populations could improve their homing and coronary vasculoprotective efficacy. Intravital imaging of the anaesthetised mouse beating heart was used to investigate the trafficking and microvascular protective effects of two clonally-derived BM-derived MSC lines, namely CD317neg MSCs-Y201 and CD317pos MSCs-Y202, cultured using conventional monolayer and 3D hanging drop methods. 3D culture consistently improved the adhesive behaviour of MSCs-Y201 to various substrates in vitro. However, it was their differential ability to reduce neutrophil events within the coronary capillaries and improve ventricular perfusion in vivo that was most remarkable. Moreover, dual therapy combined with heparin further improved the vasculoprotection afforded by 3D cultured MSCs-Y201 by also modifying platelet as well as neutrophil recruitment, which subsequently led to the greatest salvage of viable myocardium. Therapeutic benefit could mechanistically be explained by reductions in coronary endothelial oxidative stress and intercellular adhesion molecule-1 (ICAM-1)/vascular cell adhesion molecule-1 (VCAM-1) expression. However, since this was noted by both 2D and 3D cultured MSCs-Y201, therapeutic benefit is likely explained by the fact that 3D cultured MSCs-Y201 were the most potent sub-population at reducing serum levels of several pro-inflammatory cytokines. This novel study highlights the importance of not only 3D culture, but also of a specific CD317neg MSC sub-population, as being critical to realising their full coronary vasculoprotective potential in the injured heart. Since the smallest coronary blood vessels are increasingly recognised as a primary target of reperfusion injury, therapeutic interventions must be able to protect these delicate structures from inflammatory cells and maintain perfusion in the heart. We propose that relatively feasible technical modifications in a specific BM-derived MSC sub-population could achieve this.

The Stromal Vascular Fraction from Canine Adipose Tissue Contains Mesenchymal Stromal Cell Subpopulations That Show Time-Dependent Adhesion to Cell Culture Plastic Vessels

Adipose-derived mesenchymal stromal cells (MSCs) are extensively studied in both human and veterinary medicine. Their isolation is usually performed by collagenase digestion followed by filtration and removal of nonadherent tissue remnants 48 h after seeding. We observed that waste tissue fragments contain cells that adhere belatedly to the plastic. We aimed to investigate their basic properties to speculate on the possible existence of MSC subpopulations. Adipose tissue from three dogs was enzymatically digested. Three cell populations that adhered to the culture plastic 48, 96, and 144 h after seeding were obtained. After expansion, they were analyzed by flow cytometry for MSC-positive (CD90, CD44, and CD29) and -negative (CD14, MHCII, and CD45) markers as well as for endothelial, pericyte, and smooth muscle cell markers (CD31, CD146, and alpha-SMA). Furthermore, cells were assessed for viability, doubling time, and trilineage differentiation ability. No significant differences were found between the three subpopulations. As a result, this procedure has proven to be a valuable method for dramatically improving MSCs yield. As a consequence of cell recovery optimization, the amount of tissue harvested could be reduced, and the time required to obtain sufficient cells for clinical applications could be shortened. Further studies are needed to uncover possible different functional properties.

ИММУНОФЕНОТИП МЕЗЕНХИМАЛЬНЫХ СТРОМАЛЬНЫХ КЛЕТОК ПРИ РЕПЛИКАТИВНОМ СТАРЕНИИ

Mesenchymal stromal cells (MSCs), a population of progenitor cells in adult tissues, are involved in the processes of physiological tissue renewal and damage-induced regeneration. MSCs have been widely studied as regenerative medicine agents. In this regard, the tissue-specific features of MSC subpopulations should be taken into account. MSCs have many potentially beneficial properties that can alter significantly with age. The ability of a cell to respond to external signals and regulate its functional state is commonly attributed to the repertoire of receptors on the cell membrane. This article considers the surface marker expression of senescent adipose-derived MSCs (AD-MSCs). Replicative senescence was caused by long-term cultivation. An increase in the expression of CD29, CD44, CD54, CD73, CD90, and HLA-ABC on the AD-MSCs was shown. The expression of CD105 and CD51/61 did not change reliably under the experimental conditions. The revealed effects are related not only to the larger cell size or higher autofluorescence, but also to the increased number of markers per unit area of the cell surface. The detected changes may underlie a number of modifications in the properties of senescent MSCs, including migration, adhesion, and immunomodulatory and angiogenic activities.

Targeting S100A9 in the Inflammatory Myelodysplastic Hematopoietic Niche Reprograms the Functional Properties of CD271+ Mesenchymal Stromal Cells

Myelodysplastic syndromes (MDS) belong to the most common hematological malignancies in the elderly population, characterized by ineffective hematopoiesis, peripheral cytopenia and the risk of transformation into acute myeloid leukemia. An involvement of the bone marrow microenvironment (BMME) in disease initiation and progression is indisputable. The profound immune dysregulation and sustained inflammation, essentially mediated by mesenchymal stromal cells (MSCs), are further factors contributing to ineffective hematopoiesis and driving disease progression. The alarmin S100A9 is a TLR4 and CD33 ligand that acts as key player for the inflammasome activation in the MDS BM. The novel small molecular drug Tasquinimod (TASQ, Active Biotech) is a S100A9 inhibitor and has demonstrated antitumor and immunomodulatory properties in a broad range of solid tumors; however, little is known about its effects in myeloid malignancies. Here, we aim to decipher the effects of TASQ on MDS MSC subpopulations in more detail. Therefore, MSCs were isolated from BM aspirates ('asp-MSCs', n=12) or biopsies ('bp-MSCs', n=5) of patients with either low-risk MDS, CMML or age-adjusted healthy donors and were exposed to TASQ (10 µM) in vitro. Flow cytometry analysis of the MSC markers CD44, CD73, CD90, CD105, and CD146 did not show significant differences in neither asp- nor bp-MSCs after treatment. Interestingly, CD271, which is hard to detect in asp-MSCs, was decreased between 40% and 70% in bp-MSCs after TASQ treatment. This was accompanied by an inhibited expression of the checkpoint inhibitor PD-L1. The MDS BM has a higher potency for adipogenic differentiation which further supports the inflammatory conditions and the suppression of normal hematopoiesis. Treatment with TASQ resulted in about 50% inhibition of adipogenesis as demonstrated by significantly lower expression of adipocyte protein 2 in differentiated asp- and bp-MSCs. To investigate possible metabolic modulations of MSCs by TASQ, the oxygen consumption rate was measured as a readout of mitochondrial function by using the Cell Mito Stress Test (Agilent Technologies) in a XF96 analyzer. The basal and the maximal respiratory rate as well as ATP production were increased in TASQ-treated bp-MSC cultures suggesting a positive effect for their bioenergetic capacity. This was supported by detection of higher levels of glutamate dehydrogenase 1, a key enzyme in glutaminolysis converting glutamate to α-ketoglutarate for entry into the TCA cycle, and of the glutamine transporter Slc1a3 in TASQ-treated cells. NMR analyses revealed increased levels of glutamate and citrate as well as of several lipids, such as free cholesterol LDL, phospholipids LDL-6 and Apo-B LDL6 in supernatants of bp-MSCs after TASQ treatment. To investigate the impact of TASQ on CD271 expression in more detail, we sorted CD45-/CD73+/CD90+/CD271+/- bp-MSCs. Gene expression analysis by RNA sequencing of sorted MSCs revealed differential expression of several pathways, e.g. an enrichment of inflammation related molecules in the CD271+ samples, whereby TASQ could abrogate this effect. Moreover, a cytokine array analysis of culture supernatants of sorted cells showed enhanced IFNg, IL1b and IL-6 secretion of CD271+ MSCs compared to CD271- and TASQ-treated cells. To evaluate the impact on the hematopoietic support of MSCs, we analyzed CD271-sorted bp-MSC co-cultures with CD34+ hematopoietic stem and progenitor cells (HSPCs). We observed a significantly decreased number of cobblestone area forming cells (CAF-C) as well as reduced numbers of colonies (CFU) in a subsequent clonogenic assay for CD271+ MSCs, nominating this MSC subpopulation as responsible for the diminished hematopoietic support. Interestingly, the number of CAF-C and CFU-E/CFU-GM could be increased by TASQ pre-treatment of the stromal layer. In conclusion, CD271+ MSCs play a crucial role in the inflammatory MDS BMME. The inhibition by TASQ efficiently blocks adipogenic differentiation and secretion of pro-inflammatory cytokines, thereby modulating the MSC energetic profile and enhancing their potential to support hematopoiesis in vitro.

Single-cell RNA sequencing reveals different signatures of mesenchymal stromal cell pluripotent-like and multipotent populations

Somatic stem cells are advantageous research targets for understanding the properties required to maintain stemness. Human bone marrow-mesenchymal stromal cells (BM-MSCs) were separated into pluripotent-like SSEA-3(+) Muse cells (Muse-MSCs) and multipotent SSEA-3(-) MSCs (MSCs) and were subjected to single-cell RNA sequencing analysis. Compared with MSCs, Muse-MSCs exhibited higher expression levels of the p53 repressor MDM2; signal acceptance-related genes EGF, VEGF, PDGF, WNT, TGFB, INHB, and CSF; ribosomal protein; and glycolysis and oxidative phosphorylation. Conversely, MSCs had higher expression levels of FGF and ANGPT; Rho family and caveola-related genes; amino acid and cofactor metabolism; MHC class I/II, and lysosomal enzyme genes than Muse-MSCs. Unsupervised clustering further divided Muse-MSCs into two clusters stratified by the expression of cell cycle-related genes, and MSCs into three clusters stratified by the expression of cell cycle-, cytoskeleton-, and extracellular matrix-related genes. This study evaluating the differentiation ability of BM-MSC subpopulations provides intriguing insights for understanding stemness.

CD146 Defines a Mesenchymal Stromal Cell Subpopulation with Enhanced Suppressive Properties.

Mesenchymal stromal cells (MSCs) are largely known for their immune-suppressive capacity, hence, their common use in the control of unwanted inflammation. However, novel concepts related to their biology, combined with the urgent need to identify MSC subpopulations with enhanced suppressive properties, drive the search for isolation protocols optimized for clinical applications. We show, in this study, that MSCs expressing high CD146 levels exhibit altered surface expression profiles of CD44 and secrete elevated levels of interleukin (IL)-6, amongst other factors. In addition, CD146hi MSCs surpass the polyclonal parental populations in inhibiting alloreactive T cells in vitro, in both a soluble- and cell-contact-dependent manner. Despite the lack of CD146hi MSC-mediated activation of peritoneal macrophages to release the suppressive factor IL-10 in vitro, their administration in animals with graft-versus-host disease alleviates inflammation and leads to 40% survival rate up to 7 weeks post-transplantation. This pronounced inhibitory property is driven by CD146-mediated in situ efferocytosis by myeloid cells. Altogether, this study provides the impetus to adopt an isolation protocol for MSCs based on a CD146 expression profile before their therapeutic use and suggests a major role played by CD146 as a novel “eat-me” signal, capable of enhancing MSC uptake by competent phagocytes.

Multilayered omics reveal sex- and depot-dependent adipose progenitor cell heterogeneity

Single-cell RNA sequencing (scRNA-seq) has revealed that adult white adipose tissue (WAT) harbors functionally diverse subpopulations of mesenchymal stromal cells that differentially impact tissue plasticity. To date, the molecular basis of this cellular heterogeneity has not been fully defined. Here, we describe a multilayered omics approach to dissect adipose progenitor cell heterogeneity in three dimensions: progenitor subpopulation, sex, and anatomical localization. We applied state-of-the-art mass spectrometry methods to quantify 4,870 proteins in eight different stromal cell populations from perigonadal and inguinal WAT of male and female mice and acquired transcript expression levels of 15,477 genes using RNA-seq. Our data unveil molecular signatures defining sex differences in preadipocyte differentiation and identify regulatory pathways that functionally distinguish adipose progenitor subpopulations. This multilayered omics analysis, freely accessible at http://preadprofiler.net/, provides unprecedented insights into adipose stromal cell heterogeneity and highlights the benefit of complementary proteomics to support findings from scRNA-seq studies.

Single-cell transcriptomics defines Dot1L interacting partners and downstream target genes in the mouse molar dental pulp.

Although histone methyltransferases are implicated in many key developmental processes, the contribution of individual chromatin modifiers in dental tissues is not well understood. Using single-cell RNA sequencing, we examined the expression profiles of the disruptor of telomeric silencing 1-like (Dot1L) gene in the postnatal day 5 mouse molar dental pulp. Dot1L is the only known enzyme that methylates histone 3 on lysine 79, a modification associated with gene expression. Our research revealed 15 distinct clusters representing different populations of mesenchymal stromal cells (MSCs), immune cells, pericytes, ameloblasts and endothelial cells. We documented heterogeneity in gene expression across different subpopulations of MSCs, a good indicator that these stromal progenitors undergo different phases of osteogenic differentiation. Interestingly, although Dot1L was broadly expressed across all cell clusters within the molar dental pulp, our analyses indicated specific enrichment of Dot1L within two clusters of MSCs, as well as cell clusters characterized as ameloblasts and endothelial cells. Moreover, we detected Dot1L co-expression with protein interactors involved in epigenetic activation such as Setd2, Sirt1, Brd4, Isw1, Bptf and Suv39h1. In addition, Dot1L was co-expressed with Eed2, Cbx3 and Dnmt1, which encode epigenetic factors associated with gene silencing and heterochromatin formation. Dot1l was co-expressed with downstream targets of the insulin growth factor and WNT signaling pathways, as well as genes involved in cell cycle progression. Collectively, our results suggest that Dot1L may play key roles in orchestrating lineage-specific gene expression during MSC differentiation.

Functional heterogeneity of IFN-γ–licensed mesenchymal stromal cell immunosuppressive capacity on biomaterials

Mesenchymal stromal cells (MSCs) are increasingly combined with biomaterials to enhance their therapeutic properties, including their immunosuppressive function. However, clinical trials utilizing MSCs with or without biomaterials have shown limited success, potentially due to their functional heterogeneity across different donors and among different subpopulations of cells. Here, we evaluated the immunosuppressive capacity, as measured by the ability to reduce T-cell proliferation and activation, of interferon-gamma (IFN-γ)-licensed MSCs from multiple donors on fibrin and collagen hydrogels, the two most commonly utilized biomaterials in combination with MSCs in clinical trials worldwide according to ClinicalTrials.gov Variations in the immunosuppressive capacity between IFN-γ-licensed MSC donors on the biomaterials correlated with the magnitude of indoleamine-2,3-dioxygenase activity. Immunosuppressive capacity of the IFN-γ-licensed MSCs depended on the αV/α5 integrins when cultured on fibrin and on the α2/β1 integrins when cultured on collagen. While all tested MSCs were nearly 100% positive for these integrins, sorted MSCs that expressed higher levels of αV/α5 integrins demonstrated greater immunosuppressive capacity with IFN-γ licensing than MSCs that expressed lower levels of these integrins on fibrin. These findings were equivalent for MSCs sorted based on the α2/β1 integrins on collagen. These results demonstrate the importance of integrin engagement to IFN-γ licensed MSC immunosuppressive capacity and that IFN-γ-licensed MSC subpopulations of varying immunosuppressive capacity can be identified by the magnitude of integrin expression specific to each biomaterial.

Microfluidic Separation of Canine Adipose-Derived Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) are potential treatments for a variety of veterinary medical conditions. However, clinical trials have often fallen short of expectations, due in part to heterogeneity and lack of characterization of the MSCs. Identification and characterization of subpopulations within MSC cultures may improve those outcomes. Therefore, the functional heterogeneity of different-sized subpopulations of MSCs was evaluated. A high-throughput, biophysical, label-free microfluidic sorting approach was used to separate subpopulations of canine adipose-derived MSCs (Ad-MSCs) based on size for subsequent characterization, as well as to evaluate the impact of culture conditions on their functional heterogeneity. We found that culture-expanded canine Ad-MSCs comprise distinct subpopulations: larger MSCs (mean diameter of 18.6 ± 0.2 μm), smaller MSCs (mean diameter of 15.3 ± 0.2 μm), and intermediate MSCs (mean diameter of 16.9 ± 0.1 μm). In addition, proliferation characteristics, senescence, and differentiation potential of canine Ad-MSCs are also dependent on cell size. We observed that larger MSCs proliferate more slowly, senesce at earlier passages, and are inclined to differentiate into adipocytes compared with smaller MSCs. Most importantly, these size-dependent functions are also affected by the presence of serum in the culture medium, as well as time in culture. Cell surface staining for MSC-specific CD44 and CD90 antigens showed that all subpopulations of MSCs are indistinguishable, suggesting that this criterion is not relevant to define subpopulations of MSCs. Finally, transcriptome analysis showed differential gene expression between larger and smaller subpopulations of MSCs. Larger MSCs expressed genes involved in cellular senescence such as cyclin-dependent kinase inhibitor 1A and smaller MSCs expressed genes that promote cell growth [mechanistic target of rapamycin 1 (mTORC1) pathway] and cell proliferation [myelocytomatosis (myc), e2f targets]. These results suggest that different subpopulations of MSCs have specific properties. Impact statement Clinical trials of mesenchymal stromal cells (MSCs) from veterinary species have often fallen short of expectations, due in part to heterogeneity and lack of characterization of the MSCs. A high-throughput, biophysical, label-free microfluidic sorting approach was used to separate subpopulations of canine adipose-derived MSCs (Ad-MSCs) based on size for subsequent characterization. Proliferation characteristics, senescence, and differentiation potential of canine Ad-MSCs are also dependent on cell size. Cell surface staining for MSC-specific cell surface markers showed that all subpopulations of MSCs are indistinguishable, suggesting that this criterion is not relevant to define subpopulations of MSCs.

Inflammatory stromal cells in the myeloma microenvironment

Single-cell RNA sequencing identifies an inflammatory subpopulation of mesenchymal stromal cells in patients with multiple myeloma.

Hox-Positive Adult Mesenchymal Stromal Cells: Beyond Positional Identity.

Homeotic genes (Hox) are universal regulators of the body patterning process in embryogenesis of metazoans. The Hox gene expression pattern (Hox code) retains in adult tissues and serves as a cellular positional identity marker. Despite previously existing notions that the Hox code is inherent in all stroma mesenchymal cells as a whole, recent studies have shown that the Hox code may be an attribute of a distinct subpopulation of adult resident mesenchymal stromal cells (MSC). Recent evidence allows suggesting a “non-canonical” role for Hox gene expression which is associated with renewal and regeneration in postnatal organs after damage. In tissues with high regenerative capacity, it has been shown that a special cell population is critical for these processes, a distinctive feature of which is the persistent expression of tissue-specific Hox genes. We believe that in the postnatal period Hox-positive subpopulation of resident MSC may serve as a unique regenerative reserve. These cells coordinate creation and maintenance of the correct structure of the stroma through a tissue-specific combination of mechanisms. In this article, we summarize data on the role of resident MSC with a tissue-specific pattern of Hox gene expression as regulators of correct tissue reconstruction after injury.

Effect of culture medium and cell size on functional heterogeneity of canine adipose-derived mesenchymal stromal cells

Background & Aim Introduction Mesenchymal stromal cells (MSCs) are being investigated in clinical trials for treatment of a variety of veterinary conditions based on their anti-inflammatory actions and potential to induce functional tissue repair. However, outcomes of clinical trials have often fallen short of pre-clinical expectations. Heterogeneity of MSC preparations and lack of adequate characterization may be partial contributors to the variability in clinical outcomes. Purpose Identification of subpopulations within heterogenous MSC cultures may improve selection of cells with the desired functionality. Therefore, the functional heterogeneity of different subpopulations of MSCs that vary in size was evaluated, as size is hypothesized to relate to function. Methods, Results & Conclusion Methods A high-throughput, biophysical, label-free microfluidic sorting approach was used to separate subpopulations of canine adipose-derived MSCs (Ad-MSCs) based on size. Size sorted MSCs were then characterized and evaluated for the impact of culture conditions on functional heterogeneity. Results We found that larger subpopulations of MSCs with a mean diameter of ∼18 μm proliferate more slowly, senesce at earlier passages, and are inclined to form adipocytes compared to smaller MSC subpopulations (mean diameter of ∼15 μm). Most importantly, size-dependent functions are affected by culture conditions as well as time in culture. Cell surface staining for MSC-specific CD44 & CD90 antigens showed different sized subpopulations of MSCs had comparable expression levels suggesting that this criterion is not relevant to defining subpopulations of MSCs. However, transcriptome analysis showed that differently sized subpopulations of canine Ad-MSCs have distinct gene expression profiles, and culture medium and time in culture further affect gene expression. Conclusion This work provides a strategy for identifying functional subpopulations of canine MSCs based on size that could facilitate the development of quality cellular therapies through a better understanding of functional heterogeneity.

Endothelial Progenitor and Mesenchymal Stromal Cells in Newborns With Congenital Diaphragmatic Hernia Undergoing Extracorporeal Membrane Oxygenation.

Background: Endothelial progenitor (EPC) and mesenchymal stromal cells (MSC) can regenerate damaged endothelium and thereby improve pulmonary endothelial dysfunction. We do not know, how extracorporeal membrane oxygenation (ECMO) might affect EPC- and MSC-mediated regenerative pathways in patients with congenital diaphragmatic hernia (CDH). Therefore, we investigated, if ECMO support impacts EPC and MSC numbers in CDH patients.Methods: Peripheral blood mononuclear cells from newborns with ECMO-dependent (n = 18) and ECMO-independent CDH (n = 12) and from healthy controls (n = 12) were isolated. The numbers of EPC and MSC were identified by flowcytometry. Serum levels of vascular endothelial growth factor (VEGF) and angiopoietin (Ang)-2 were determined.Results: EPC and MSC were elevated in newborns with CDH. ECMO-dependent infants had higher EPC subpopulation counts (2,1–7,6-fold) before treatment compared to ECMO-independent infants. In the disease course, EPC and MSC subpopulation counts in ECMO-dependent infants were lower than before ECMO initiation. During ECMO, VEGF serum levels were significantly reduced (by 90.5%) and Ang2 levels significantly increased (by 74.8%).Conclusions: Our data suggest that ECMO might be associated with a rather impaired mobilization of EPC and MSC and with a depression of VEGF serum levels in newborns with CDH.

A Biophysically Defined MSC Subpopulation for Enhanced Homing and Immunosuppression

Aims:Systemically administered bone marrow (BM) mesenchymal stromal cells (MSCs) are potentially efficacious cell-therapies for the treatment of graft-vs-host disease (GVHD) or auto-immune complications. For infused MSCs to successfully resolve immune reactions, effective homing and biodistribution to target tissues are important pre-requisites and the delivered MSCs should also be biologically primed. However, retrospective studies have now demonstrated that MSCs, when ex vivoexpanded to reach numbers relevant for human use, are a heterogeneous cell population consisting of subpopulations with different functionalities. The overall non-uniform cellular properties result in unreliable biodistribution and immunosuppressive functions. A systematic study of expanded MSCs to discern useful subpopulations is hindered by the lack of unique surface markers. In lieu of immunophenotype, we hypothesized and verified that biophysical properties of MSCs can be a useful strategy for isolating unique subpopulations. Here, we will describe a clinically relevant biophysical strategy for deriving a primed MSC subpopulation that is equipped with the necessary properties for homing and immune suppression. Methods:MSCs were derived from the healthy BM of patients aged 21-40 years and culture expanded for ~10-15 population doublings. To study putative MSC subtypes, we employed multivariate biophysical analyses of cell size, stiffness and nuclear fluctuations to identify subpopulations and a label-free microfluidic approach that takes advantage of unique biophysical cell traits was developed for cell sorting. This enabled correlation of these biophysical quantitative measures with biological attributes as well as in vitroand in vivofunctionality. For this study, the different biophysically derived MSC subpopulations were subjected to functional testing for tissue homing, engraftment as well as immune suppression. Results:A multivariate analysis of MSCs using cell diameter, membrane stiffness and nuclear membrane fluctuations is predictive of at least four subpopulations in expanded MSCs: uni-potent osteogenic progenitors, bi-potent osteochondro progenitors, uni-potent adipogenic progenitors and tri-potent progenitors. These subpopulations are distinct in their transcriptome and differentiation potential but no surface marker reliably distinguishes them apart. Importantly, the tri-potent subpopulation possessed higher transcript and protein levels of alpha4, cx3cr1, cxcr4 and podxl, compared to the other subpopulations (~2X to ~4X). In functional assays for homing and engraftment, tri-potent progenitors showed increased chemotaxis towards SDF1a(31-43% increase) and secretome from commercial cancer cell lines (MDA-MB-231/435, 26-31% increase) as well as TNFastimulated HUVECs (27-55% increase). In MSC rolling in vitroassays on TNFastimulated HUVECs, the tri-potent progenitors exhibited reduced velocities (113±45 mm/s vs >350 mm/s compared to other subpopulations), indicating a greater level of interactions with the stimulated HUVECs, and finally leading to a greater number of cell arrest on the HUVEC layer (3-5 fold increase). In vivobiodistribution experiments against subcutaneously implanted MDA-MB-231/435 cancers as well as sub-lethally irradiated mice further demonstrated a greater level of homing and engraftment towards sites of inflammation (eg. implanted tumors, myeloablated BM, gut). In vitro, tri-potent progenitors were also stronger IDO, PGE2and TGFbproducers following stimulation with INFgand was able to suppress PHA stimulated CD8 T-cell proliferation most potently (76±18% suppression vs <48%). Conclusions: These results demonstrate that heterogeneity in expanded MSCs subpopulations could lead to inconsistencies in their overall properties. Newer biophysical technologies have the potential for isolating useful subpopulations defined by key quality attributes for specific forms of MSC-therapy; these capabilities may advance MSC-based therapy for regenerative medicine. Specifically, we demonstrate a strategy for isolating the MSC subpopulation that is primed towards homing and immune suppression functions. Further validation of this subpopulation against other MSC subpopulations in animal models of GVHD or auto-immune diseases will be necessary for next translational steps. Disclosures No relevant conflicts of interest to declare.