Mesenchymal Lineages Research Articles

Inflammation, Bone Healing and Osteonecrosis: From Bedside to Bench.

Osteonecrosis of the epiphyseal and metaphyseal regions of major weight-bearing bones of the extremities is a condition that is associated with local death of bone cells and marrow in the afflicted compartment. Chronic inflammation is a prominent feature of osteonecrosis. If the persistent inflammation is not resolved, this process will result in progressive collapse and subsequent degenerative arthritis. In the pre-collapse stage of osteonecrosis, attempt at joint preservation rather than joint replacement in this younger population with osteonecrosis is a major clinical objective. In this regard, core decompression, with/without local injection of bone marrow aspirate concentrate (BMAC), is an accepted and evidence-based method to help arrest the progression and improve the outcome of early-stage osteonecrosis. However, some patients do not respond favorably to this treatment. Thus, it is prudent to consider strategies to mitigate chronic inflammation concurrent with addressing the deficiencies in osteogenesis and vasculogenesis in order to save the affected joint. Interestingly, the processes of inflammation, osteonecrosis, and bone healing are highly inter-related. Therefore, modulating the biological processes and crosstalk among cells of the innate immune system, the mesenchymal stem cell-osteoblast lineage and others are important to providing the local microenvironment for resolution of inflammation and subsequent repair. This review summarizes the clinical and biologic principles associated with osteonecrosis and provides potential cutting-end strategies for modulating chronic inflammation and facilitating osteogenesis and vasculogenesis using local interventions. Although these studies are still in the preclinical stages, it is hoped that safe, efficacious, and cost-effective interventions will be developed to save the host’s natural joint.

EWSR1/FUS–CREB fusions define a distinctive malignant epithelioid neoplasm with predilection for mesothelial-lined cavities

Gene fusions constitute pivotal driver mutations often encoding aberrant chimeric transcription factors. However, an increasing number of gene fusion events have been shown not to be histotype specific and shared among different tumor types, otherwise completely unrelated clinically or phenotypically. One such remarkable example of chromosomal translocation promiscuity is represented by fusions between EWSR1 or FUS with genes encoding for CREB-transcription factors family (ATF1, CREB1, and CREM), driving the pathogenesis of various tumor types spanning mesenchymal, neuroectodermal, and epithelial lineages. In this study, we investigate a group of 13 previously unclassified malignant epithelioid neoplasms, frequently showing an epithelial immunophenotype and marked predilection for the peritoneal cavity, defined by EWSR1/FUS–CREB fusions. There were seven females and six males, with a mean age of 36 (range 9–63). All except three cases occurred intra-abdominally, including one each involving the pleural cavity, upper, and lower limb soft tissue. All tumors showed a predominantly epithelioid morphology associated with cystic or microcystic changes and variable lymphoid cuffing either intermixed or at the periphery. All except one case expressed EMA and/or CK, five were positive for WT1, while being negative for melanocytic and other mesothelioma markers. Nine cases were confirmed by various RNA-sequencing platforms, while in the remaining four cases the gene rearrangements were detected by FISH. Eleven cases showed the presence of CREM-related fusions (EWSR1–CREM, 7; FUS–CREM, 4), while the remaining two harbored EWSR1–ATF1 fusion. Clinically, seven patients presented with and/or developed metastases, confirming a malignant biologic potential. Our findings expand the spectrum of tumors associated with CREB-related fusions, defining a novel malignant epithelioid neoplasm with an immunophenotype suggesting epithelial differentiation. This entity appears to display hybrid features between angiomatoid fibrous histiocytoma (cystic growth and lymphoid cuffing) and mesothelioma (peritoneal/pleural involvement, epithelioid phenotype, and cytokeratin and WT1 co-expression).

Mesenchyme-derived IGF2 is a major paracrine regulator of pancreatic growth and function.

The genetic mechanisms that determine the size of the adult pancreas are poorly understood. Imprinted genes, which are expressed in a parent-of-origin-specific manner, are known to have important roles in development, growth and metabolism. However, our knowledge regarding their roles in the control of pancreatic growth and function remains limited. Here we show that many imprinted genes are highly expressed in pancreatic mesenchyme-derived cells and explore the role of the paternally-expressed insulin-like growth factor 2 (Igf2) gene in mesenchymal and epithelial pancreatic lineages using a newly developed conditional Igf2 mouse model. Mesenchyme-specific Igf2 deletion results in acinar and beta-cell hypoplasia, postnatal whole-body growth restriction and maternal glucose intolerance during pregnancy, suggesting that the mesenchyme is a developmental reservoir of IGF2 used for paracrine signalling. The unique actions of mesenchymal IGF2 are demonstrated by the absence of any discernible growth or functional phenotypes upon Igf2 deletion in the developing pancreatic epithelium. Additionally, increased IGF2 levels specifically in the mesenchyme, through conditional Igf2 loss-of-imprinting or Igf2r deletion, leads to pancreatic acinar overgrowth. Furthermore, ex-vivo exposure of primary acinar cells to exogenous IGF2 activates AKT, a key signalling node, and increases their number and amylase production. Based on these findings, we propose that mesenchymal Igf2, and perhaps other imprinted genes, are key developmental regulators of adult pancreas size and function.

Trb3 controls mesenchymal stem cell lineage fate and enhances bone regeneration by scaffold-mediated local gene delivery

Aberrant lineage commitment of mesenchymal stem cells (MSCs) in marrow contributes to abnormal bone formation due to reduced osteogenic and increased adipogenic potency. While several major transcriptional factors associated with lineage differentiation have been found during the last few decades, the molecular switch for MSC fate determination and its role in skeletal regeneration remains largely unknown, limiting creation of effective therapeutic approaches. Tribbles homolog 3 (Trb3), a member of tribbles family pseudokinases, is known to exert diverse roles in cellular differentiation. Here, we investigated the reciprocal role of Trb3 in the regulation of osteogenic and adipogenic differentiation of MSCs in the context of bone formation, and examined the mechanisms by which Trb3 controls the adipo-osteogenic balance. Trb3 promoted osteoblastic commitment of MSCs at the expense of adipocyte differentiation. Mechanistically, Trb3 regulated cell-fate choice of MSCs through BMP/Smad and Wnt/β-catenin signals. Importantly, in vivo local delivery of Trb3 using a novel gelatin-conjugated caffeic acid-coated apatite/PLGA (GelCA-PLGA) scaffold stimulated robust bone regeneration and inhibited fat-filled cyst formation in rodent non-healing mandibular defect models. These findings demonstrate Trb3-based therapeutic strategies that favor osteoblastogenesis over adipogenesis for improved skeletal regeneration and future treatment of bone-loss disease. The distinctive approach implementing a scaffold-mediated local gene transfer may further broaden the translational use of targeting specific therapeutic gene related to lineage commitment for clinical bone treatment.

Regulation of TAZ by DEPTOR controls mesenchymal progenitors lineage commitment in response to PTH1R signaling

Regulation of TAZ by DEPTOR controls mesenchymal progenitors lineage commitment in response to PTH1R signaling

Assessing the Resident Progenitor Cell Population and the Vascularity of the Adult Human Meniscus

To identify, characterize, and compare the resident progenitor cell populations within the red-red, red-white, and white-white (WW) zones of freshly harvested human cadaver menisci and to characterize the vascularity of human menisci using immunofluorescence and 3-dimensional (3D) imaging. Fresh adult human menisci were harvested from healthy donors. Menisci were enzymatically digested, mononuclear cells isolated, and characterized using flow cytometry with antibodies against mesenchymal stem cell surface markers (CD105, CD90, CD44, and CD29). Cells were expanded in culture, characterized, and compared with bone marrow-derived mesenchymal stem cells. Trilineage differentiation potential of cultured cells was determined. Vasculature of menisci was mapped in 3D using a modified uDisco clearing and immunofluorescence against vascular markers CD31, lectin, and alpha smooth muscle actin. There were no significant differences in the clonogenicity of isolated cells between the 3 zones. Flow cytometry showed presence of CD44+CD105+CD29+CD90+ cells in all 3 zones with high prevalence in the WW zone. Progenitors from all zones were found to be potent to differentiate to mesenchymal lineages. Larger vessels in the red-red zone of meniscus were observed spanning toward red-white, sprouting to smaller arterioles and venules. CD31+ cells were identified in all zones using the 3D imaging and co-localization of additional markers of vasculature (lectin and alpha smooth muscle actin) was observed. The presence of resident mesenchymal progenitors was evident in all 3 meniscal zones of healthy adult donors without injury. In addition, our results demonstrate the presence of vascularization in the WW zone. The existence of progenitors and presence of microvasculature in the WW zone of the meniscus suggests the potential for repair and biologic augmentation strategies in that zone of the meniscus in young healthy adults. Further research is necessary to fully define the functionality of the meniscal blood supply and its implications for repair.

Diagnosis and management of tropomyosin receptor kinase (TRK) fusion sarcomas: expert recommendations from the World Sarcoma Network.

Sarcomas are a heterogeneous group of malignancies with mesenchymal lineage differentiation. The discovery of neurotrophic tyrosine receptor kinase (NTRK) gene fusions as tissue-agnostic oncogenic drivers has led to new personalized therapies for a subset of patients with sarcoma in the form of tropomyosin receptor kinase (TRK) inhibitors. NTRK gene rearrangements and fusion transcripts can be detected with different molecular pathology techniques, while TRK protein expression can be demonstrated with immunohistochemistry. The rarity and diagnostic complexity of NTRK gene fusions raise a number of questions and challenges for clinicians. To address these challenges, the World Sarcoma Network convened two meetings of expert adult oncologists and pathologists and subsequently developed this article to provide practical guidance on the management of patients with sarcoma harboring NTRK gene fusions. We propose a diagnostic strategy that considers disease stage and histologic and molecular subtypes to facilitate routine testing for TRK expression and subsequent testing for NTRK gene fusions.

Transcriptome profiles acquired during cell expansion and licensing validate mesenchymal stromal cell lineage genes

BackgroundMesenchymal stromal cells (MSCs) are rapidly advancing as commercial therapeutics. However, there are still no adequate tools to validate the identity of MSCs and support standardization of MSC-based products. Currently accepted metrics include cell surface marker profiling and tri-lineage differentiation assays, neither of which is definitive. Transcript profiling represents a cost- and time-effective approach amenable to MSC manufacturing processes. Two independent labs recently reported non-overlapping MSC-specific transcriptomic signatures of 489 and 16 genes.MethodsHere, we interrogated our repository of transcriptome data to determine whether routine culture manipulations including cell expansion and immune activation affect expression of the reported MSC lineage genes. These data sets comprise 4 donor populations of human umbilical cord (UC) MSCs serially cultured from cryopreservation thaw through pre-senescence, and 3 donor populations each of naïve UC and bone marrow (BM) MSCs and licensed by 3 different cytokines.ResultsOverall, 437 of 456 proposed signature genes assessed in these data sets were reliably expressed, representing an enduring lineage profile in 96% agreement with the previous studies. Serial passaging resulted in the downregulation of 3 signature genes, and one was silenced. Cytokine stimulation downregulated expression of 16 signature genes, and 3 were uniformly silenced in one or the other MSC type. Fifteen additional genes were unreliably detected, independent of culture manipulation.ConclusionThese results validate and refine the proposed transcriptomic tools for reliable identification of MSCs after isolation through cell expansion and after inflammatory activation. We propose a 24-gene signature to support standardized and accessible MSC characterization.

Abstract 340: ABI1 and androgen dependence in prostate cancer

Abstract Background: Androgen independence is the most challenging problem in the treatment of advanced prostate cancer. Although most patients with early-stage prostate cancer can be cured with surgical and/or radiation treatment, the remainder of patients ultimately recur with metastatic disease upon development of resistance to anti-androgen therapy. Epithelial mesenchymal transition (EMT) and lineage plasticity has been proposed as key mechanisms underlying resistance in treatment-induced prostate cancer with neuroendocrine features (NEPC). There has been a rise in treatment-induced NEPC with increased use of anti-androgen therapies. ABI1 regulates EMT by suppressing FYN-STAT3 activation downstream from noncanonical WNT pathway, but the interaction of ABI1 with androgen pathway is not known. We sought to determine the effect of neoadjuvant hormone treatment (NHT) on ABI1 expression levels in different tumor types. Methods: Here we analyzed ABI1 expression (IHC) in tissue microarray containing 75 tumors with no prior treatment, 198 patients subjected to NHT prior to surgery and 39 castration resistant tumors (total n=307). Androgen-induced ABI1 expression was determined upon R1881 treatment by Western blotting and immunofluorescence in hormone- sensitive LNCaP cells. Results: ABI1 expression was significantly downregulated in tumors from patients undergoing NHT(p<0.05). ABI1 expression was significantly increased in CRPC tumors developed while on hormone therapy compared to ABI1 expression in tumors undergoing NHT (p<0.001), or with NEPC (p<0.001). Consistently with these observations, ABI1 expression was upregulated upon treatment of LNCaP cells with R1881 and downregulated by treatment of cells with enzalutamide. Conclusion: ABI1 is an androgen sensitive gene. ABI1 expression is increased in CRPC, most likely secondary to androgen receptor reactivation. It is subsequently downregulated in NEPC, hence, we hypothesize that this may be a key mechanism driving the NEPC phenotype through promoting EMT. Further understanding of the role ABI1 may play in this transformation will provide a more mechanistic understanding of prostate cancer progression and maylead to improved targeted treatment options. Citation Format: Leszek Kotula, Baylee A. Porter, Xiang Li, Maria A. Ortiz, Rakesh Khanna, Fan Zhang, Sonia H. Kung, Ladan Fazli, Martin E. Gleave, Gennady Bratslavsky, Jeffrey S. Ross. ABI1 and androgen dependence in prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 340.

Isolation of mesenchymal stem cells from Pap smear samples.

ObjectiveExploiting their ability to differentiate into mesenchymal lineages like cartilage, bone, fat, and muscle, and to elicit paracrine effects, mesenchymal stem cells (MSCs) are widely used in clinical settings to treat tissue injuries and autoimmune disorders. One of accessible sources of MSC is the samples used for Papanicolaou (Pap) test, which is a cervical screening method for detecting potentially pre-cancerous and cancerous alterations in the cervical cells and to diagnose genetic abnormalities in fetuses. This study aimed to identify and isolate the stem cells from Pap smear samples collected from pregnant women, and to trace the origin of these cells to maternal or fetal tissue, and characterize their stem cell properties.MethodsTo investigate the possibility and efficiency of establishing MSC lines from the Pap smear samples, we were able to establish 6 cell lines from Pap smear samples from 60 pregnant women at different stages of gestation.ResultsThe 3 cell lines randomly selected among the 6 established in this study, displayed high proliferation rates, several characteristics of MSCs, and the capacity to differentiate into adipocytes, osteocytes, and chondrocytes. Our study identified that the stem cell lines obtainable from Pap smear sampling were uterine cervical stromal cells (UCSCs) and had 10% efficiency of establishment.Conclusion Despite their low efficiency of establishment, human UCSCs from Pap smear samples can become a simple, safe, low-cost, and donor-specific source of MSCs for stem cell therapy and regenerative medicine.

Endothelial monolayer disruption in bioprosthetic heart valve as a trigger of primary tissue failure

Aim . To study the surface and cellular composition of non-calcified bioprosthetic heart valve (BHV) leaflets with varying degrees of structural deterioration to determine the possible mechanisms of primary tissue failure development. Materials and methods . An examination of six bioprosthetic heart valves (KemCor and PeriCor) extracted from mitral position due to the structural valve deterioration was performed. The structure of BHV leaflets was studied by hematoxylin – eosin staining and immunohistochemistry assay (with the following indicators – CD3, T lymphocytes; CD20, B lymphocytes; CD31, mature endothelial cells; CD34, endothelial progenitor cells; CD68, monocytes/macrophages; vimentin, mesenchymal cells; α-smooth muscle actin, vascular smooth muscle cells). Results . The degree of disruption of BHV leaflets in primary tissue failure differed significantly: relatively intact samples with the intact endothelial monolayer, areas with impairment of the surface layers (minimal and moderate damage) and areas with the spread of destruction into the extracellular matrix of the leaflet (expressed degeneration) were determined. Endothelial cells (monolayer with preserved or impaired integrity), macrophages, smooth muscle cells and other mesenchymal lineage cells were identified in BHV. T- and B-lymphocytes were not detected in the BHV leaflets. Conclusions . A characteristic feature of structurally deteriorated BHVs is impairment of endothelial monolayer integrity in areas of degraded extracellular matrix. In contrast to other types of bioprosthetic dysfunctions, structural valve deterioration was characterized by the absence of lymphocyte infiltration. Therefore, we suppose that endothelial mololayer injury is a trigger of structural BHV deterioration.

Incorporation of the first and second heart fields and prospective fate of the straight heart tube via in vivo labeling of chicken embryos.

Recent discoveries of at least two heart fields and dynamic nature of cardiac development as well as controversies regarding the participation of heart fields in development of different heart structures led us to investigate the dynamics of incorporation of the first and second heart fields and prospective fate of the straight heart tube by labeling chicken embryos in vivo with the fluorescent lipophilic dye DiI. The cephalic and caudal limits of the anterior and posterior segments of the straight heart tube were labeled in two groups of embryos. Labels were tracked along the “C,” “S,” and “U” loops up to the tetracavitary or mature heart (n = 30 embryos/group; torsion and looping stage). To determine whether the atria and atrioventricular canal are derived from the first heart field the straight heart tube was cultured in vitro and immunodetection of Sox-9 and troponin I was performed to identify the mesenchymal and myocardial lineages respectively. Proliferating cell nuclear antigen (PCNA) immunodetection was used to determine the involvement of cell proliferation in heart tube development during torsion and looping. Embryological constitution of the straight heart tube and heart looping (C, S, and U) were not consistent with current descriptions. In fact, right ventricle precursors were absent in the straight heart tube derived from the first heart field. During torsion and looping, the cephalic segment of the straight heart tube gradually shifted into the heart tube until it was located at the myocardial interventricular septum in the tetracavitary heart. In contrast, the caudal segment of the straight heart tube was elongated and remodeled to become the first heart field derived left ventricle and the proximal part of the ventricular inlets. The ventricular outflows, right ventricle, distal part of the ventricular inlets, and atria developed from the second heart field.

Single-cell RNA sequencing reveals profibrotic roles of distinct epithelial and mesenchymal lineages in pulmonary fibrosis.

Pulmonary fibrosis (PF) is a form of chronic lung disease characterized by pathologic epithelial remodeling and accumulation of extracellular matrix (ECM). To comprehensively define the cell types, mechanisms, and mediators driving fibrotic remodeling in lungs with PF, we performed single-cell RNA sequencing of single-cell suspensions from 10 nonfibrotic control and 20 PF lungs. Analysis of 114,396 cells identified 31 distinct cell subsets/states. We report that a remarkable shift in epithelial cell phenotypes occurs in the peripheral lung in PF and identify several previously unrecognized epithelial cell phenotypes, including a KRT5- /KRT17 + pathologic, ECM-producing epithelial cell population that was highly enriched in PF lungs. Multiple fibroblast subtypes were observed to contribute to ECM expansion in a spatially discrete manner. Together, these data provide high-resolution insights into the complexity and plasticity of the distal lung epithelium in human disease and indicate a diversity of epithelial and mesenchymal cells contribute to pathologic lung fibrosis.

Hydrogel Properties May Influence Mesenchymal Stem Cell Lineage Progression Through Modulating GAPDH Activity

Cell traction forces, biochemical signals, and cell metabolism are each known to regulate mesenchymal stem cell (MSC) differentiation. Biomaterials have therefore been designed to manipulate cell traction force (via their viscoelasticity and adhesion ligands) to guide MSC fate decisions. Similarly, the type and density of biochemical signals presented by a material have been tailored to influence MSC differentiation. However, the potential impact of biomaterial properties on regulating MSC differentiation through modulating cell metabolism is relatively unstudied. Here, we present data indicating that hydrogel elastic modulus and mesh size regulate MSC differentiation in part through modulating the activity of the metabolic enzyme glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). Toward this end, we first confirm that the differentiation profile of MSCs cultured in 2D on highly elastic, covalently crosslinked poly(ethylene glycol) diacrylate (PEGDA) hydrogels differs substantially from that of MSCs encapsulated within the same hydrogel formulations, indicating a dependence in 3D on a variable(s) beyond elastic modulus. Further results indicate that the GAPDH activity of MSCs in 3D hydrogels is a function of elastic modulus and mesh size, suggesting that GAPDH activity may be one of these variables. Studies in 2D supported a positive correlation between hydrogel elastic modulus and GAPDH activity. Additionally, inhibition of GAPDH activity on 2D surfaces induced alterations in the profiles of key differentiation markers, indicating that GAPDH activity can impact lineage progression. Cumulatively, these findings suggest that the potential impact of hydrogel properties on cell metabolism should be considered when evaluating biomaterial-driven MSC differentiation. Cell traction forces, biochemical signals, and cell metabolism are each known to regulate mesenchymal stem cell (MSC) differentiation. Biomaterials have therefore been designed to manipulate cell traction force (via their viscoelasticity and adhesion ligands) to guide MSC fate decisions. Similarly, the type and density of biochemical signals presented by a material have been tailored to influence MSC differentiation. However, the impact of biomaterial properties on regulating MSC differentiation through modulating cell metabolism is relatively unstudied. Here, we present data indicating that hydrogel elastic modulus and mesh size regulate MSC differentiation in part through modulating the activity of the metabolic enzyme glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).

Ischemia-Induced Multipotent Stem Cells Isolated from Stroke Patients Exhibit Higher Neurogenic Differentiation Potential than Bone Marrow-Derived Mesenchymal Stem Cells.

Perivascular areas of the brain harbor multipotent stem cells. We recently demonstrated that after a stroke, brain pericytes exhibit features of multipotent stem cells. Moreover, these ischemia-induced multipotent stem cells (iSCs) are present within ischemic areas of the brain of patients diagnosed with stroke. Although increasing evidence shows that iSCs have traits similar to those of mesenchymal stem cells (MSCs), the phenotypic similarities and differences between iSCs and MSCs remain unclear. In this study, we used iSCs extracted from stroke patients (h-iSCs) and compared their neurogenic potential with that of human MSCs (h-MSCs) in vitro. Microarray analysis, fluorescence-activated cell sorting, immunohistochemistry, and multielectrode array were performed to compare the characteristics of h-iSCs and h-MSCs. Although h-iSCs and h-MSCs had similar gene expression profiles, the percentage expressing the neural stem/progenitor cell marker nestin was significantly higher in h-iSCs than in h-MSCs. Consistent with these findings, h-iSCs, but not h-MSCs, differentiated into electrophysiologically functional neurons. In contrast, although both h-iSCs and h-MSCs were able to differentiate into several mesodermal lineages, including adipocytes, osteocytes, and chondrocytes, the potential of h-iSCs to differentiate into adipocytes and osteocytes was relatively low. These results suggest that compared with h-MSCs, h-iSCs predominantly exhibit neural rather than mesenchymal lineages. In addition, these results indicate that h-iSCs have the potential to repair the injured brain of patients with stroke by directly differentiating into neuronal lineages.

Plasticity in Ovarian Cancer: The Molecular Underpinnings and Phenotypic Heterogeneity

Cellular plasticity, by large, is the ability through which cells morph into new phenotypic identity by trading-off with the previous one. This phenomenon has been observed in the normal and tumor cells in a very similar fashion. Occurrence of cellular plasticity, in malignancies like epithelial ovarian carcinoma (EOC) are well known but highly debated in terms of origin of the tumor for the inherent heterogeneity across subtypes. EOC has been termed as a clinically challenging malady for its subversive nature against chemotherapy. The management of ovarian cancer is mostly hindered by relapse with recalcitrance towards primary chemotherapy regimen e.g. platinum–taxol combination. Also, late detection preceded by peritoneal metastasis poses another challenge to the treatment. Underlying both these aspects of the disease, tumor heterogeneity turns out as the most critical factor. In the light of heterogeneity that can be across patients (inter-tumoral) and/or within a tumor (intra-tumoral), ovarian carcinoma is a multifactorial ailment. The governing factors behind this heterogeneity are—diverse genetic landscape among subtypes of EOC; the presence of cancer stem cell (CSC) niche and inherent plasticity of the cancer cells themselves. Apart from the well-studied mechanisms of plasticity, there are several emerging molecular players like lncRNAs, Hippo pathway and also phenomena like dedifferentiation of non-CSC neoplastic cells ,and transdifferentiation of CSCs. Here, we present an overview of the current knowledge on the evolution of EOC through cellular plasticity with emphasis on the three major aspects namely, subtype-specific genetic diversity; ovarian CSC and cancer cell duality between epithelial and mesenchymal lineages.

Sensory nerves regulate mesenchymal stromal cell lineage commitment by tuning sympathetic tones.

The sensory nerve was recently identified as being involved in regulation of bone mass accrual. We previously discovered that prostaglandin E2 (PGE2) secreted by osteoblasts could activate sensory nerve EP4 receptor to promote bone formation by inhibiting sympathetic activity. However, the fundamental units of bone formation are active osteoblasts, which originate from mesenchymal stromal/stem cells (MSCs). Here, we found that after sensory denervation, knockout of the EP4 receptor in sensory nerves, or knockout of COX-2 in osteoblasts, could significantly promote adipogenesis and inhibit osteogenesis in adult mice. Furthermore, injection of SW033291 (a small molecule that locally increases the PGE2 level) or propranolol (a beta blocker) significantly promoted osteogenesis and inhibited adipogenesis. This effect of SW033291, but not propranolol, was abolished in conditional EP4-KO mice under normal conditions or in the bone repair process. We conclude that the PGE2/EP4 sensory nerve axis could regulate MSC differentiation in bone marrow of adult mice.

Epigenetic Features of Human Perinatal Stem Cells Redefine Their Stemness Potential.

Human perinatal stem cells (SCs) can be isolated from fetal annexes without ethical or safety limitations. They are generally considered multipotent; nevertheless, their biological characteristics are still not fully understood. The aim of this study was to investigate the pluripotency potential of human perinatal SCs as compared to human induced pluripotent stem cells (hiPSCs). Despite the low expression of the pluripotent factors NANOG, OCT4, SOX2, and C-KIT in perinatal SC, we observed minor differences in the promoters DNA-methylation profile of these genes with respect to hiPSCs; we also demonstrated that in perinatal SCs miR-145-5p had an inverse trend in comparison to these stemness markers, suggesting that NANOG, OCT4, and SOX2 were regulated at the post-transcriptional level. The reduced expression of stemness markers was also associated with shorter telomere lengths and shift of the oxidative metabolism between hiPSCs and fetal annex-derived cells. Our findings indicate the differentiation ability of perinatal SCs might not be restricted to the mesenchymal lineage due to an epigenetic barrier, but other regulatory mechanisms such as telomere shortening or metabolic changes might impair their differentiation potential and challenge their clinical application.

The elephant in the lung: Integrating lineage-tracing, molecular markers, and single cell sequencing data to identify distinct fibroblast populations during lung development and regeneration

During lung development, the mesenchyme and epithelium are dependent on each other for instructive morphogenic cues that direct proliferation, cellular differentiation and organogenesis. Specification of epithelial and mesenchymal cell lineages occurs in parallel, forming cellular subtypes that guide the formation of both transitional developmental structures and the permanent architecture of the adult lung. While epithelial cell types and lineages have been relatively well-defined in recent years, the definition of mesenchymal cell types and lineage relationships has been more challenging. Transgenic mouse lines with permanent and inducible lineage tracers have been instrumental in identifying lineage relationships among epithelial progenitor cells and their differentiation into distinct airway and alveolar epithelial cells. Lineage tracing experiments with reporter mice used to identify fibroblast progenitors and their lineage trajectories have been limited by the number of cell specific genes and the developmental timepoint when the lineage trace was activated. In this review, we discuss major developmental mesenchymal lineages, focusing on time of origin, major cell type, and other lineage derivatives, as well as the transgenic tools used to find and define them. We describe lung fibroblasts using function, location, and molecular markers in order to compare and contrast cells with similar functions. The temporal and cell-type specific expression of fourteen “fibroblast lineage” genes were identified in single-cell RNA-sequencing data from LungMAP in the LGEA database. Using these lineage signature genes as guides, we clustered murine lung fibroblast populations from embryonic day 16.5 to postnatal day 28 (E16.5-PN28) and generated heatmaps to illustrate expression of transcription factors, signaling receptors and ligands in a temporal and population specific manner.

Ethanol Inhibits Mesenchymal Stem Cell Osteochondral Lineage Differentiation Due in Part to an Activation of Forkhead Box Protein O-Specific Signaling.

During bone fracture repair, resident mesenchymal stem cells (MSCs) differentiate into chondrocytes, to form a cartilaginous fracture callus, and osteoblasts, to ossify the collagen matrix. Our laboratory previously reported that alcohol administration led to decreased cartilage formation within the fracture callus of rodents and this effect was mitigated by postfracture antioxidant treatment. Forkhead box protein O (FoxO) transcription factors are activated in response to intracellular reactive oxygen species (ROS), and alcohol has been shown to increase ROS. Activation of FoxOs has also been shown to inhibit canonical Wnt signaling, a necessary pathway for MSC differentiation. These findings have led to our hypothesis that alcohol exposure decreases osteochondrogenic differentiation of MSCs through the activation of FoxOs. Primary rat MSCs were treated with ethanol (EtOH) and assayed for FoxO expression, FoxO activation, and downstream target expression. Next, MSCs were differentiated toward osteogenic or chondrogenic lineages in the presence of 50mM EtOH and alterations in osteochondral lineage marker expression were determined. Lastly, osteochondral differentiation experiments were repeated with FoxO1/3 knockdown or with FoxO1/3 inhibitor AS1842856 and osteochondral lineage marker expression was determined. EtOH increased the expression of FoxO3a at mRNA and protein levels in primary cultured MSCs. This was accompanied by an increase in FoxO1 nuclear localization, FoxO1 activation, and downstream catalase expression. Moreover, EtOH exposure decreased expression of osteogenic and chondrogenic lineage markers. FoxO1/3 knockdown restored proosteogenic and prochondrogenic lineage marker expression in the presence of 50mM EtOH. However, FoxO1/3 inhibitor only restored proosteogenic lineage marker expression. These data show that EtOH has the ability to inhibit MSC differentiation, and this ability may rely, at least partially, on the activation of FoxO transcription factors.