Heterogeneous Population Of Stromal Cells Research Articles

Hypoxia promotes an inflammatory phenotype of fibroblasts in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an extensive fibroinflammatory stroma and often experiences conditions of insufficient oxygen availability or hypoxia. Cancer-associated fibroblasts (CAF) are a predominant and heterogeneous population of stromal cells within the pancreatic tumor microenvironment. Here, we uncover a previously unrecognized role for hypoxia in driving an inflammatory phenotype in PDAC CAFs. We identify hypoxia as a strong inducer of tumor IL1ɑ expression, which is required for inflammatory CAF (iCAF) formation. Notably, iCAFs preferentially reside in hypoxic regions of PDAC. Our data implicate hypoxia as a critical regulator of CAF heterogeneity in PDAC.

Profound abnormalities in thymic epithelial cells in Rag1 hypomorphic mice: implications for immune reconstitution after stem cell transplantation

Abstract Thymic epithelial cells (TEC) are a heterogeneous population of stromal cells which orchestrate T cell development and selection. Although recent reports highlighted TEC complexity, their developmental origin, hierarchy and differentiation still remain ill-defined. We compared TEC distribution and gene expression in wild-type (WT) mice and in mice carrying Rag1 hypomorphic mutations (Rag1mut) observed in patients with immunedeficiency and immune dysregulation. Single cell RNA-seq analysis of TECs isolated from Rag1mut mice revealed an excess of cortical TECs (cTECs). Medullary TECs (mTECs), albeit reduced in number, showed a similar distribution in previously described mature subsets. To address whether TEC abnormalities in Rag1mut mice might result from a developmental block, we compared them to TECs from neonate WT mice. Indeed, we observed a similar TEC distribution in adult Rag1mut mice and newborn WT mice, although we identified peculiar features in cTEC subsets of Rag1mut mice. Transplantation of WT hematopoietic stem cells (HSC) into Rag1mut mice was able not only to correct thymocyte development, but also to revert TEC phenotype to normalcy. However, in a competitive transplantation setting where 10% of WT and 90% mutant HSCs were used, only partial restoration of thymocyte subsets was observed, associated with incomplete rescue of TEC phenotype. In summary, we show that abnormalities of TEC composition in Rag1 mutant mice correlate with impaired development of thymocytes, highlighting the role of lymphostromal cross-talk in TEC maturation. Moreover, these data point to the need to achieve full donor chimerism after HSC transplantation in order to rescue the thymic compartment and prevent defects of immune tolerance. This research was supported by the Intramural Research Program of the NIAID, NIH

Deciphering the temporal heterogeneity of cancer-associated fibroblast subpopulations in breast cancer

BackgroundCancer-associated fibroblasts (CAFs) comprise a heterogeneous population of stromal cells within the tumour microenvironment. CAFs exhibit both tumour-promoting and tumour-suppressing functions, making them exciting targets for improving cancer treatments. Careful isolation, identification, and characterisation of CAF heterogeneity is thus necessary for ex vivo validation and future implementation of CAF-targeted strategies in cancer.MethodsMurine 4T1 (metastatic) and 4T07 (poorly/non-metastatic) orthotopic triple negative breast cancer tumours were collected after 7, 14, or 21 days. The tumours were analysed via flow cytometry for the simultaneous expression of six CAF markers: alpha smooth muscle actin (αSMA), fibroblast activation protein alpha (FAPα), platelet derived growth factor receptor alpha and beta (PDGFRα and PDGFRβ), CD26/DPP4 and podoplanin (PDPN). All non-CAFs were excluded from the analysis using a lineage marker cocktail (CD24, CD31, CD45, CD49f, EpCAM, LYVE-1, and TER-119). In total 128 murine tumours and 12 healthy mammary fat pads were analysed.ResultsWe have developed a multicolour flow cytometry strategy based on exclusion of non-CAFs and successfully employed this to explore the temporal heterogeneity of freshly isolated CAFs in the 4T1 and 4T07 mouse models of triple-negative breast cancer. Analysing 128 murine tumours, we identified 5–6 main CAF populations and numerous minor ones based on the analysis of αSMA, FAPα, PDGFRα, PDGFRβ, CD26, and PDPN. All markers showed temporal changes with a distinct switch from primarily PDGFRα+ fibroblasts in healthy mammary tissue to predominantly PDGFRβ+ CAFs in tumours. CD26+ CAFs emerged as a large novel subpopulation, only matched by FAPα+ CAFs in abundance.ConclusionWe demonstrate that multiple subpopulations of CAFs co-exist in murine triple negative breast cancer, and that the abundance and dynamics for each marker differ depending on tumour type and time. Our results form the foundation needed to isolate and characterise specific CAF populations, and ultimately provide an opportunity to therapeutically target specific CAF subpopulations.

Mesenchymal Stromal Cells in Viral Infections: Implications for COVID-19.

Mesenchymal stromal cells (MSCs) constitute a heterogeneous population of stromal cells with immunomodulatory and regenerative properties that support their therapeutic use. MSCs isolated from many tissue sources replicate vigorously in vitro and maintain their main biological properties allowing their widespread clinical application. To date, most MSC-based preclinical and clinical trials targeted immune-mediated and inflammatory diseases. Nevertheless, MSCs have antiviral properties and have been used in the treatment of various viral infections in the last years. Here, we revised in detail the biological properties of MSCs and their preclinical and clinical applications in viral diseases, including the disease caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection (COVID-19). Notably, rapidly increasing numbers of MSC-based therapies for COVID-19 have recently been reported. MSCs are theoretically capable of reducing inflammation and promote lung regeneration in severe COVID-19 patients. We critically discuss the rationale, advantages and disadvantages of MSC-based therapies for viral infections and also specifically for COVID-19 and point out some directions in this field. Finally, we argue that MSC-based therapy may be a promising therapeutic strategy for severe COVID-19 and other emergent respiratory tract viral infections, beyond the viral infection diseases in which MSCs have already been clinically applied. Graphical Electronic supplementary materialThe online version of this article (10.1007/s12015-020-10032-7) contains supplementary material, which is available to authorized users.

Single cell RNAseq analysis reveals profound abnormalities in the distribution and diversity of thymic epithelial cells in Rag1 mutant mice

Abstract The thymus contains a heterogeneous population of stromal cells which orchestrate T cell development and selection. Recent works highlighted the complexity of thymic stromal cells, and medullary thymic epithelial cells (mTECs) in particular. However, the developmental origin, hierarchy, and function of these subpopulations remain ill-defined. Moreover, the characterization of cortical TECs (cTECs) has been largely restricted to the adult thymus. Here, we compared TEC distribution and gene expression in wild-type (WT) and in mice carrying Rag1 hypomorphic mutations observed in patients with immune deficiency and immune dysregulation. Single cell RNA-seq analysis of TECs isolated from adult Rag1 mutant mice revealed an excess of cTECs, which segregated in different clusters. The mTEC compartment, albeit reduced, showed a similar distribution of previously described subsets (mTEC I–IV), suggesting perturbation of mTEC development rather than mTEC differentiation into functional subsets. To address whether such abnormalities of cTEC and mTEC abundance and subset distribution in Rag1 mutant mice may reflect defects in TEC development, we extended scRNA-seq analysis to TECs from WT mice of neonatal age. A similar pattern was observed in TECs of adult Rag1 mutant mice and WT mice of neonatal age, indicating altered TEC development when Rag1 activity is perturbed. This defect correlates with a decreased number of double-positive thymocytes, suggesting that impaired lymphostromal cross-talk in the thymus of Rag1 mutant mice (and patients with similar defects) is associated with abnormalities of TEC composition which may contribute to altered immune tolerance that is often observed in these conditions.

Dose-dependent Preservation of beta-cell function in type i diabetes by mesenchymal stromal cells

Background & Aim Background Wharton's jelly derived mesenchymal stromal cells (WJMSCs) are a heterogeneous stromal cell population characterized as MSCs as per the ISCT's minimal criteria. This cell source possesses strong immunomodulatory properties, is reliably isolated from easily accessible tissue, and can be expanded in vitro to generate therapeutic doses of cell product (under the classification of an advanced therapeutic medicinal product [ATMP]). Methods, Results & Conclusion Methods ProTrans®, an allogeneic WJMSC product pooled from five donors in each batch, and expanded in vitro for a maximum of three passages, was manufactured under good manufacturing practice conditions, and evaluated for safety and efficacy to preserve beta-cell function in a phase I/IIa clinical trial of adult (18-40 years), newly diagnosed patients with type 1 diabetes (Clinicaltrials.gov identifier: NCT03406585). Under the first phase of the trial, an open, dose-escalating study was performed in 9 male patients, using three different doses of ProTrans®, 25 (low), 100 (medium) and 200 (high) million cells. Cells were delivered in a single intravenous (IV) infusion and patients evaluated 12 months post-treatment. Results No adverse effects related to ProTrans® infusion were observed in any of the treatment groups. No human leukocyte antigen (HLA) immunization occurred. Mixed meal tolerance tests were performed prior to treatment and after one year. C-peptide responses, as measured as area under the curve, decreased in subjects receiving the lowest dose of 25 million cells, while subjects treated with medium or high doses maintained their C-peptide levels during the first year (P=0.014 comparing low to medium doses and P=0.011 comparing low to high doses). No significant difference was seen in C-peptide responses between medium and high dose therapy. Conclusions We conclude that Protrans® MSCs can be safely administered IV in the three tested doses, and that treatment in the two higher doses may exhibit the beneficial effect of preserving C-peptide for at least a year in adult patients newly diagnosed with type 1 diabetes. This study is currently continuing as a blinded phase I/IIa trial including both male and female patients, to further substantiate our safety data and establish efficacy of therapy.

Immunoevolution of mouse pancreatic organoid isografts from preinvasive to metastatic disease

Pancreatic ductal adenocarcinoma (PDA) has a highly immunosuppressive microenvironment, which is contributed by the complex interaction between cancer cells and a heterogeneous population of stromal cells. Therefore, facile and trackable models are needed for integrative and dynamic interrogation of cancer-stroma interaction. Here, we tracked the immunoevolution of PDA in a genetically-defined transplantable model of mouse pancreatic tumour organoids that recapitulates the progression of the disease from early preinvasive lesions to metastatic carcinomas. We demonstrated that organoid-derived isografts (ODI) can be used as a biological source of biomarkers (NT5E, TGFB1, FN1, and ITGA5) of aggressive molecular subtypes of human PDA. In ODI, infiltration from leukocytes is an early event during progression of the disease as observed for autochthonous models. Neoplastic progression was associated to accumulation of Maf+ macrophages, which inversely correlated with CD8+ T cells infiltration. Consistently, levels of MAF were enriched in human PDA subtypes characterized by abundance of macrophage-related transcripts and indicated poor patients’ survival. Density of MAF+ macrophages was higher in human PDA tissues compared to preinvasive lesions. Our results suggest that ODIs represent a suitable system for genotypic-immunophenotypic studies and support the hypothesis of MAF+ macrophages as a prominent immunosuppressive population in PDA.

Abstract 4495: A CD10+GPR77+ subset of cancer-associated fibroblasts promotes cancer formation and chemoresistance by sustaining cancer stem cells

Abstract Carcinoma-associated fibroblasts (CAFs) are an abundant and heterogeneous population of stromal cells in the tumor microenvironment critically involved in cancer progression. However, the lack of effective tools to isolate CAF subsets has prevented a comprehensive functional characterization of CAFs as well as their therapeutic potentials. Here, we demonstrate that two cell-surface molecules, CD10 and GPR77, specifically define a new CAF subset correlated with chemoresistance and poor survival in multiple cohorts of breast and lung cancer patients. CD10+GPR77+ CAFs promote tumor formation and chemoresistance by providing a survival niche for the cancer stem cells (CSCs). Mechanistically the functions of the CAF subset are driven by persistent NF-κB activation via p65 phosphorylation and acetylation, which is maintained by complement signaling via GPR77, a C5a receptor. Furthermore, CD10+GPR77+ CAFs are crucial for the successful engraftment of patient-derived xenografts (PDXs) in vivo and targeting these CAFs with a neutralizing anti-GPR77 antibody abolishes tumor formation and restores tumor chemosensitivity. This discovery represents the first time a functional CAF subset is identified using specific surface markers, which will greatly facilitate future investigations of the tumor microenvironment and suggests that targeting the CD10+GPR77+ CAF subset could be an effective therapeutic strategy against CSCs-driven solid tumors. Citation Format: Erwei Song. A CD10+GPR77+ subset of cancer-associated fibroblasts promotes cancer formation and chemoresistance by sustaining cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4495.

Targeting Bone Marrow Mesenchymal Stromal Cells Using Cre-Recombinase Transgenes

The bone marrow microenvironment contains hematopoietic niches that regulate the proliferation, differentiation, and trafficking of hematopoietic stem/progenitors cells (HSPCs). These hematopoietic niches are comprised of a heterogeneous population of stromal cells that include, endothelial cells, osteoblasts, CXCL12-abundant reticular (CAR) cells, mesenchymal stem cells (MSCs), arteriolar pericytes, and sympathetic nerves. Emerging data suggest that specific stromal populations may regulate distinct types of HPSCs. Thus, it is important to have validated approaches to interrogate and target specific stromal cell populations. Prior studies have shown that Prx1-Cre, Osx-Cre, Lepr-Cre, and Nes-Cre broadly target mesenchymal stromal cells in the bone marrow. Here, we rigorously define the stromal cell populations targeted by two Cre-transgenes that are commonly used to target osteolineage cells (Ocn-Cre, and Dmp1-Cre) and introduce a new Cre-transgene (Tagln-Cre) that efficiently targets bone marrow pericytes. For each Cre-transgene, we performed lineage mapping using ROSA26Ai9/Ai9 mice, in which cells that have undergone Cre-mediated recombination express tdTomato. In some cases, we further crossed these mice to introduce the Cxcl12gfp transgene, which can be used to define GFP-bright CAR cells. Immunostaining of bone sections and flow cytometry were used to define the target stromal cell population(s) in these mice. Osteocalcin (Bglap, Ocn) is primarily expressed in mature osteoblasts. Accordingly, Ocn-Cre is widely used to specifically target osteoblasts. However, our lineage mapping studies show that Ocn-Cre targets not only all osteoblasts, but also 72 ± 4.0% of CAR cells. Ocn-Cre also targets a subset of NG2+ arteriolar pericytes. Dentin matrix acidic phosphoprotein 1 (Dmp1) is expressed primarily in osteocytes, and Dmp1-Cre has been widely used to specifically target osteocytes. However, we show that Dmp1-Cre also efficiently targets endosteal osteoblasts and approximately 40% of CAR cells. To target bone marrow pericytes, we tested several Cre-transgenes, ultimately focusing on Tagln-Cre. Transgelin (Tagln, SM22a) is broadly expressed in pericytes, smooth muscle cells, and cardiomyocytes. Lineage-mapping studies show that Tagln-Cre targets all arteriolar and venous sinusoidal pericytes in the bone marrow. It also targets osteoblasts and 75 ± 5.2% of CAR cells. There are several recent studies that have ascribed specific functions to osteoblasts or osteocytes based on targeting using Ocn-Cre or Dmp1-Cre, respectively. In light of our data, these conclusions need to be re-evaluated. Ocn-Cre, Dmp1-Cre, and Tagln-Cre each target a subset of CAR cells. Studies are underway to determine whether these CAR subsets have unique expression profiles and functions. Finally, Talgn-Cre represents a new tool for investigators in the field to efficiently target bone marrow pericytes. Disclosures No relevant conflicts of interest to declare.

The hematopoietic stem cell niche in homeostasis and disease

AbstractThe bone marrow microenvironment contains a heterogeneous population of stromal cells organized into niches that support hematopoietic stem cells (HSCs) and other lineage-committed hematopoietic progenitors. The stem cell niche generates signals that regulate HSC self-renewal, quiescence, and differentiation. Here, we review recent studies that highlight the heterogeneity of the stromal cells that comprise stem cell niches and the complexity of the signals that they generate. We highlight emerging data that stem cell niches in the bone marrow are not static but instead are responsive to environmental stimuli. Finally, we review recent data showing that hematopoietic niches are altered in certain hematopoietic malignancies, and we discuss how these alterations might contribute to disease pathogenesis.

TGF-β signaling in stromal cells acts upstream of FGF-10 to regulate epithelial stem cell growth in the adult lung

Tissue resident mesenchymal stromal cells (MSCs) contribute to tissue regeneration through various mechanisms, including the secretion of trophic factors that act directly on epithelial stem cells to promote epithelialization. However, MSCs in tissues constitute a heterogeneous population of stromal cells and different subtypes may have different functions. In this study we show that CD166neg and CD166pos lung stromal cells have different proliferative and differentiative potential. CD166neg lung stromal cells exhibit high proliferative potential with the capacity to differentiate along the lipofibroblastic and myofibroblastic lineages, whereas CD166pos lung stromal cells have limited proliferative potential and are committed to the myofibroblastic lineage. Moreover, we show that CD166pos lung stromal cells do not share the same epithelial-supportive capacity as their CD166neg counterparts, which support the growth of lung epithelial stem cell (EpiSPC) colonies in vitro. In addition, ex vivo expansion of lung stromal cells also resulted in the loss of epithelial-supportive capacity, which could be reinstated by inhibition of the TGF-β signaling pathway. We show that epithelial-supportive capacity correlated with the level of FGF-10 expression and the reactivation of several lung development-associated genes. In summary, these studies suggest that TGF-β signaling in stromal cells acts upstream of FGF-10 to regulate epithelial stem cell growth in the adult lung.

Implication of Tumor Microenvironment in Chemoresistance: Tumor-Associated Stromal Cells Protect Tumor Cells from Cell Death

Tumor development principally occurs following the accumulation of genetic and epigenetic alterations in tumor cells. These changes pave the way for the transformation of chemosensitive cells to chemoresistant ones by influencing the uptake, metabolism, or export of drugs at the cellular level. Numerous reports have revealed the complexity of tumors and their microenvironment with tumor cells located within a heterogeneous population of stromal cells. These stromal cells (fibroblasts, endothelial or mesothelial cells, adipocytes or adipose tissue-derived stromal cells, immune cells and bone marrow-derived stem cells) could be involved in the chemoresistance that is acquired by tumor cells via several mechanisms: (i) cell–cell and cell–matrix interactions influencing the cancer cell sensitivity to apoptosis; (ii) local release of soluble factors promoting survival and tumor growth (crosstalk between stromal and tumor cells); (iii) direct cell-cell interactions with tumor cells (crosstalk or oncologic trogocytosis); (iv) generation of specific niches within the tumor microenvironment that facilitate the acquisition of drug resistance; or (v) conversion of the cancer cells to cancer-initiating cells or cancer stem cells. This review will focus on the implication of each member of the heterogeneous population of stromal cells in conferring resistance to cytotoxins and physiological mediators of cell death.

Clinical Application of Mesenchymal Stem Cells in the Treatment and Prevention of Graft-versus-Host Disease

Mesenchymal stem cells (MSCs) represent a heterogeneous population of stromal cells with pluripotent mesenchymal differentiation potential. They have been found to have immunosuppressive properties and the ability to modulate angiogenesis and endogenous tissue repair by in vitro and animal studies. Clinical trials have examined the utility of these cells in autoimmune and inflammatory conditions. In particular, in allogeneic hematopoietic stem cell transplant (HSCT), multiple studies have been conducted to explore the use of MSC to treat acute and chronic graft-versus-host disease (GVHD) and for cotransplantation with HSCT to promote HSC engraftment and prevent GVHD. We review here the results of these studies and discuss some challenges of this treatment modality in this disease setting.

Potential of mesenchymal stem cells for the therapy of autoimmune diseases

Mesenchymal stem cells (MSCs) are a heterogeneous population of stromal cells that are usually isolated from the bone marrow and differentiate into cells of mesodermal lineage. MSCs exert immunosuppressive activities by suppressing T- and B-cell proliferation, dampening the generation of mature myeloid dendritic cells, and inhibiting the proliferation, cytokine production and cytotoxic activity of natural killer cells. Their immunomodulatory features, together with their tissue-trophic properties, make MSCs good candidates to treat autoimmune disorders (ADs). Different preclinical models of ADs clearly demonstrate the beneficial effects of MSCs on injured tissues by inhibiting immune inflammation and promoting tissue repair through trophic and anti-apoptotic mechanisms. Although these results pave the way toward the design of clinical trials with MSCs in AD patients, studies published so far are few in number. However, it appears from these studies that administration of MSCs is safe and feasible.

4 Adhesion molecules in haemopoiesis

In the adult mammal, haemopoiesis is restricted to the extravascular compartment of the bone marrow (BM) where primitive haemopoietic stem cells (HSC) and their clonogenic progeny develop in intimate contiguity with a heterogeneous population of stromal cells that comprise the haemopoietic micro-environment (HM). Although the importance of cellular interactions between primitive haemopoietic progenitor cells (HPC) and marrow stromal cells is well established, precise definition of the nature of many of these interactions at the molecular level is lacking and remains an objective of fundamental importance to understanding of haemopoietic regulation. Current data suggest that a wide variety of cell surface molecules representing several adhesion molecule superfamilies, including integrins, selectins, sialomucins and the immunoglobulin gene superfamily, are involved in supporting cell-cell and cell-extracellular matrix (ECM) interactions. These diverse CAM-ligand interactions, rather than simply serving to initiate and maintain contact between HPC and stromal cells and ECM components, also have an additional, more direct role in controlling the growth and development of primitive haemopoietic cells.

A method for separating cultured preadipocytes according to their density: application to stromal cells from overfed suckling rats.

The stroma vascular fraction of adipose tissue consists of a heterogeneous cell population; not all the cells in this compartment undergo adipose conversion in primary culture. A density gradient centrifugation procedure was used to separate cultured cells on the basis of their triglyceride content. This method was applied to both stroma vascular cells from rat adipose tissue and to a 3T3 F442A preadipose cell line as a reference. Comparison of the results obtained from these two cell types suggests that this separation procedure can lead to a quantification of adipose differentiation in the heterogeneous stromal cell population. Separation procedures were applied to cultured stromal cells derived from young rats during the onset of nutritional obesity induced by overfeeding in early life. Results show that early overfeeding induced an increase in the stromal cell differentiation capacity which is expressed in vitro.