Homogeneous Cell Population Research Articles

Biological characteristics of a sub-population of cancer stem cells from two triple-negative breast tumour cell lines

Triple-negative breast tumours (TNBTs) make up 15–20% of all breast tumours. There is no treatment for them, and the role that cancer stem cells (CSCs) have in carcinogenesis is still unclear, so finding markers and therapeutic targets in CSC exosomes requires these cells to exist as a homogeneous cell population. The objective of this work was to determine differences in ultrastructural morphology, proliferative capacity, and mouse-xenotransplantation characteristics of the MDA-MB-231 and MDA-MB-436 TNBT cell lines with the CD44high/CD24low phenotype in order to study their exosomes. The results show that the CD44high/CD24low MBA-MB-231 cells had a population doubling time of 41.56 h, compared to 44.79 h in the MDA-MB-436 cell line. After magnetic immunoseparation, 18.75% and 14.56% of the stem cell population of the MDA-MB-231 and MDA-MB-436 cell lines, respectively, were of the CD44high/CD24low phenotype, which were expanded to reach purities of 80.4% and 87.6%. The same expanded lineage in both cell lines was shown to possess the pluripotency markers Nanog and Oct4. Under a scanning electron microscope, the CD44high/CD24low lineage of the MBA-MD-231 cell line formed groups of more interconnected cells than this lineage of the MBA-MD-436 line. A total of 16% of the mice inoculated with the CD44high/CD24low lineage of either cell line presented tumours of the breast, lung, and submandibular ganglia, in whose tissues variable numbers of inoculated cells were found 30 days post-inoculation. By magnetic immunoselection, it was possible to isolate in similar quantities and characterize, expand, and xenotransplant the CD44high/CD24low lineage of the MDA-MB-231 and MDA-MB-436 cell lines. The former cell line has greater proliferative capacity, the two lines differ under scanning electron microscopy in how they intercommunicate, and both cell lines induce new tumours in mice and persist at least 30 days post-inoculation in the transplanted animal so their exosomes would also be different.

Single-cell RNA sequencing of cultured human endometrial CD140b+CD146+ perivascular cells highlights the importance of in vivo microenvironment

BackgroundEndometrial mesenchymal-like stromal/stem cells (eMSCs) have been proposed as adult stem cells contributing to endometrial regeneration. One set of perivascular markers (CD140b&CD146) has been widely used to enrich eMSCs. Although eMSCs are easily accessible for regenerative medicine and have long been studied, their cellular heterogeneity, relationship to primary counterpart, remains largely unclear.MethodsIn this study, we applied 10X genomics single-cell RNA sequencing (scRNA-seq) to cultured human CD140b+CD146+ endometrial perivascular cells (ePCs) from menstrual and secretory endometrium. We also analyzed publicly available scRNA-seq data of primary endometrium and performed transcriptome comparison between cultured ePCs and primary ePCs at single-cell level.ResultsTranscriptomic expression-based clustering revealed limited heterogeneity within cultured menstrual and secretory ePCs. A main subpopulation and a small stress-induced subpopulation were identified in secretory and menstrual ePCs. Cell identity analysis demonstrated the similar cellular composition in secretory and menstrual ePCs. Marker gene expression analysis showed that the main subpopulations identified from cultured secretory and menstrual ePCs simultaneously expressed genes marking mesenchymal stem cell (MSC), perivascular cell, smooth muscle cell, and stromal fibroblast. GO enrichment analysis revealed that genes upregulated in the main subpopulation enriched in actin filament organization, cellular division, etc., while genes upregulated in the small subpopulation enriched in extracellular matrix disassembly, stress response, etc. By comparing subpopulations of cultured ePCs to the publicly available primary endometrial cells, it was found that the main subpopulation identified from cultured ePCs was culture-unique which was unlike primary ePCs or primary endometrial stromal fibroblast cells.ConclusionIn summary, these data for the first time provides a single-cell atlas of the cultured human CD140b+CD146+ ePCs. The identification of culture-unique relatively homogenous cell population of CD140b+CD146+ ePCs underscores the importance of in vivo microenvironment in maintaining cellular identity.

A top-down measure of gene-to-gene coordination for analyzing cell-to-cell variability

Recent technological advances, such as single-cell RNA sequencing (scRNA-seq), allow the measurement of gene expression profiles of individual cells. These expression profiles typically exhibit substantial variations even across seemingly homogeneous populations of cells. Two main different sources contribute to this measured variability: actual differences between the biological activity of the cells and technical measurement errors. Analysis of the biological variability may provide information about the underlying gene regulation of the cells, yet distinguishing it from the technical variability is a challenge. Here, we apply a recently developed computational method for measuring the global gene coordination level (GCL) to systematically study the cell-to-cell variability in numerical models of gene regulation. We simulate ‘biological variability’ by introducing heterogeneity in the underlying regulatory dynamic of different cells, while ‘technical variability’ is represented by stochastic measurement noise. We show that the GCL decreases for cohorts of cells with increased ‘biological variability’ only when it is originated from the interactions between the genes. Moreover, we find that the GCL can evaluate and compare—for cohorts with the same cell-to-cell variability—the ratio between the introduced biological and technical variability. Finally, we show that the GCL is robust against spurious correlations that originate from a small sample size or from the compositionality of the data. The presented methodology can be useful for future analysis of high-dimensional ecological and biochemical dynamics.

Sex-Specific Isolation and Propagation of Human Premeiotic Fetal Germ Cells and Germ Cell-Like Cells.

The second trimester of human development is marked by asynchronous gonadal development hampering the isolation of homogenous populations of early and late fetal germ cells (FGCs). We evaluated the feasibility of using surface markers TNAP, PDPN, EPCAM and ITGA6 to isolate FGCs as well as human primordial germ cell-like cells (hPGCLCs) derived from embryonic stem cells (hESCs) from both sexes by fluorescence-activated cell sorting (FACS). Our results suggest that a combination of TNAP and PDPN was sufficient to separate populations of premeiotic FGCs and hPGCLCs in both sexes. This combination of antibodies also proved efficient in separating ‘mitotic’ from ‘retinoic-acid responsive’ female FGCs. Furthermore, we report that the differentiation efficiency of TNAP+PDPN+ hPGCLCs from hESCs was sex-independent, but the ability to propagate differed considerably between the sexes. In contrast to male, female hPGCLCs retained their characteristics and exhibited robust colony-forming ability when cultured for five days in medium containing LIF, forskolin and FGF2. We conclude that marked sex differences exist in the isolation and propagation of human FGCs and hPGCLCs. Our study provides novel insights relevant for the optimization of in vitro gametogenesis in humans.

Hematopoietic Stem Cells: Nature and Niche Nurture.

Like all cells, hematopoietic stem cells (HSCs) and their offspring, the hematopoietic progenitor cells (HPCs), are highly sociable. Their capacity to interact with bone marrow niche cells and respond to environmental cytokines orchestrates the generation of the different types of blood and immune cells. The starting point for engineering hematopoiesis ex vivo is the nature of HSCs, and a longstanding premise is that they are a homogeneous population of cells. However, recent findings have shown that adult bone marrow HSCs are really a mixture of cells, with many having lineage affiliations. A second key consideration is: Do HSCs “choose” a lineage in a random and cell-intrinsic manner, or are they instructed by cytokines? Since their discovery, the hematopoietic cytokines have been viewed as survival and proliferation factors for lineage committed HPCs. Some are now known to also instruct cell lineage choice. These fundamental changes to our understanding of hematopoiesis are important for placing niche support in the right context and for fabricating an ex vivo environment to support HSC development.

Cell-specific epigenetic changes in atherosclerosis.

Atherosclerosis is a disease of large and medium arteries that can lead to life-threatening cerebrovascular and cardiovascular consequences such as heart failure and stroke and is a major contributor to cardiovascular-related mortality worldwide. Atherosclerosis development is a complex process that involves specific structural, functional and transcriptional changes in different vascular cell populations at different stages of the disease. The application of single-cell RNA sequencing (scRNA-seq) analysis has discovered not only disease-related cell-specific transcriptomic profiles but also novel subpopulations of cells once thought as homogenous cell populations. Vascular cells undergo specific transcriptional changes during the entire course of the disease. Epigenetics is the instruction-set-architecture in living cells that defines and maintains the cellular identity by regulating the cellular transcriptome. Although different cells contain the same genetic material, they have different epigenomic signatures. The epigenome is plastic, dynamic and highly responsive to environmental stimuli. Modifications to the epigenome are driven by an array of epigenetic enzymes generally referred to as writers, erasers and readers that define cellular fate and destiny. The reversibility of these modifications raises hope for finding novel therapeutic targets for modifiable pathological conditions including atherosclerosis where the involvement of epigenetics is increasingly appreciated. This article provides a critical review of the up-to-date research in the field of epigenetics mainly focusing on in vivo settings in the context of the cellular role of individual vascular cell types in the development of atherosclerosis.

Instructive roles of astrocytes in hippocampal synaptic plasticity: neuronal activity-dependent regulatory mechanisms.

In the adult hippocampus, synaptic plasticity is important for information processing, learning, and memory encoding. Astrocytes, the most common glial cells, play a pivotal role in the regulation of hippocampal synaptic plasticity. While astrocytes were initially described as a homogenous cell population, emerging evidence indicates that in the adult hippocampus, astrocytes are highly heterogeneous and can differentially respond to changes in neuronal activity in a subregion‐dependent manner to actively modulate synaptic plasticity. In this review, we summarize how local neuronal activity changes regulate the interactions between astrocytes and synapses, either by modulating the secretion of gliotransmitters and synaptogenic proteins or via contact‐mediated signaling pathways. In turn, these specific responses induced in astrocytes mediate the interactions between astrocytes and neurons, thus shaping synaptic communication in the adult hippocampus. Importantly, the activation of astrocytic signaling is required for memory performance including memory acquisition and recall. Meanwhile, the dysregulation of this signaling can cause hippocampal circuit dysfunction in pathological conditions, resulting in cognitive impairment and neurodegeneration. Indeed, reactive astrocytes, which have dysregulated signaling associated with memory, are induced in the brains of patients with Alzheimer's disease (AD) and transgenic mouse model of AD. Emerging technologies that can precisely manipulate and monitor astrocytic signaling in vivo enable the examination of the specific actions of astrocytes in response to neuronal activity changes as well as how they modulate synaptic connections and circuit activity. Such findings will clarify the roles of astrocytes in hippocampal synaptic plasticity and memory in health and disease.

Natural Killer Cells Generated From Human Induced Pluripotent Stem Cells Mature to CD56brightCD16+NKp80+/-In-Vitro and Express KIR2DL2/DL3 and KIR3DL1.

The differentiation of human induced pluripotent stem cells (hiPSCs) into T and natural killer (NK) lymphocytes opens novel possibilities for developmental studies of immune cells and in-vitro generation of cell therapy products. In particular, iPSC-derived NK cells gained interest in adoptive anti-cancer immunotherapies, since they enable generation of homogenous populations of NK cells with and without genetic engineering that can be grown at clinical scale. However, the phenotype of in-vitro generated NK cells is not well characterized. NK cells derive in the bone marrow and mature in secondary lymphoid tissues through distinct stages from CD56brightCD16- to CD56dimCD16+ NK cells that represents the most abandoned population in peripheral blood. In this study, we efficiently generated CD56+CD16+CD3- NK lymphocytes from hiPSC and characterized NK-cell development by surface expression of NK-lineage markers. Hematopoietic priming of hiPSC resulted in 31.9% to 57.4% CD34+CD45+ hematopoietic progenitor cells (HPC) that did not require enrichment for NK lymphocyte propagation. HPC were further differentiated into NK cells on OP9-DL1 feeder cells resulting in high purity of CD56brightCD16- and CD56brightCD16+ NK cells. The output of generated NK cells increased up to 40% when OP9-DL1 feeder cells were inactivated with mitomycine C. CD7 expression could be detected from the first week of differentiation indicating priming towards the lymphoid lineage. CD56brightCD16-/+ NK cells expressed high levels of DNAM-1, CD69, natural killer cell receptors NKG2A and NKG2D, and natural cytotoxicity receptors NKp46, NKp44, NKp30. Expression of NKp80 on 40% of NK cells, and a perforin+ and granzyme B+ phenotype confirmed differentiation up to stage 4b. Killer cell immunoglobulin-like receptor KIR2DL2/DL3 and KIR3DL1 were found on up to 3 and 10% of mature NK cells, respectively. NK cells were functional in terms of cytotoxicity, degranulation and antibody-dependent cell-mediated cytotoxicity.

Efficient differentiation of human embryonic stem cells to retinal pigment epithelium under defined conditions

Age-related macular degeneration (AMD) is a highly prevalent form of blindness caused by loss death of cells of the retinal pigment epithelium (RPE). Transplantation of pluripotent stem cell (PSC)-derived RPE cells is considered a promising therapy to regenerate cell function and vision.ObjectiveThe objective of this study is to develop a rapid directed differentiation method for production of RPE cells from PSC which is rapid, efficient, and fully defined and produces cells suitable for clinical use.DesignA protocol for cell growth and differentiation from hESCs was developed to induce differentiation through screening small molecules which regulated a primary stage of differentiation to the eyefield progenitor, and then, a subsequent set of molecules to drive differentiation to RPE cells. Methods for cell plating and maintenance have been optimized to give a homogeneous population of cells in a short 14-day period, followed by a procedure to support maturation of cell function.ResultsWe show here the efficient production of RPE cells from human embryonic stem cells (hESCs) using small molecules in a feeder-free system using xeno-free/defined medium. Flow cytometry at day 14 showed ~ 90% of cells expressed the RPE markers MITF and PMEL17. Temporal gene analysis confirmed differentiation through defined cell intermediates. Mature hESC-RPE cell monolayers exhibited key morphological, molecular, and functional characteristics of the endogenous RPE.ConclusionThis study identifies a novel cell differentiation process for rapid and efficient production of retinal RPE cells directly from hESCs. The described protocol has utility for clinical-grade cell production for human therapy to treat AMD.

A sub-population of Dictyostelium discoideum cells shows extremely high sensitivity to cAMP for directional migration

The chemotaxis of Dictysotelium discoideum cells in response to a chemical gradient of cyclic adenosine 3′,5′-monophosphate (cAMP) was studied using a newly designed microfluidic device. The device consists of 800 cell-sized channels in parallel, each 4 μm wide, 5 μm high, and 100 μm long, allowing us to prepare the same chemical gradient in all channels and observe the motility of 500–1000 individual cells simultaneously. The percentage of cells that exhibited directed migration was determined for various cAMP concentrations ranging from 0.1 pM to 10 μM. The results show that chemotaxis was highest at 100 nM cAMP, consistent with previous observations. At concentrations as low as 10 pM, about 16% of cells still exhibited chemotaxis, suggesting that the receptor occupancy of only 6 cAMP molecules/cell can induce chemotaxis in very sensitive cells. At 100 pM cAMP, chemotaxis was suppressed due to the self-production and secretion of intracellular cAMP induced by extracellular cAMP. Overall, systematic observations of a large number of individual cells under the same chemical gradients revealed the heterogeneity of chemotaxis responses in a genetically homogeneous cell population, especially the existence of a sub-population with extremely high sensitivity for chemotaxis.

Enrichment of Skeletal Stem Cells from Human Bone Marrow Using Spherical Nucleic Acids.

Human bone marrow (BM)-derived stromal cells contain a population of skeletal stem cells (SSCs), with the capacity to differentiate along the osteogenic, adipogenic, and chondrogenic lineages, enabling their application to clinical therapies. However, current methods to isolate and enrich SSCs from human tissues remain, at best, challenging in the absence of a specific SSC marker. Unfortunately, none of the current proposed markers alone can isolate a homogeneous cell population with the ability to form bone, cartilage, and adipose tissue in humans. Here, we have designed DNA-gold nanoparticles able to identify and sort SSCs displaying specific mRNA signatures. The current approach demonstrates the significant enrichment attained in the isolation of SSCs, with potential therein to enhance our understanding of bone cell biology and translational applications.

Process development and safety evaluation of ABCB5+ limbal stem cells as advanced-therapy medicinal product to treat limbal stem cell deficiency

BackgroundWhile therapeutic success of the limbal tissue or cell transplantation to treat severe cases of limbal stem cell (LSC) deficiency (LSCD) strongly depends on the percentage of LSCs within the transplanted cells, prospective LSC enrichment has been hampered by the intranuclear localization of the previously reported LSC marker p63. The recent identification of the ATP-binding cassette transporter ABCB5 as a plasma membrane-spanning marker of LSCs that are capable of restoring the cornea and the development of an antibody directed against an extracellular loop of the ABCB5 molecule stimulated us to develop a novel treatment strategy based on the utilization of in vitro expanded allogeneic ABCB5+ LSCs derived from human cadaveric limbal tissue.MethodsWe developed and validated a Good Manufacturing Practice- and European Pharmacopeia-conform production and quality-control process, by which ABCB5+ LSCs are derived from human corneal rims, expanded ex vivo, isolated as homogenous cell population, and manufactured as an advanced-therapy medicinal product (ATMP). This product was tested in a preclinical study program investigating the cells’ engraftment potential, biodistribution behavior, and safety.ResultsABCB5+ LSCs were reliably expanded and manufactured as an ATMP that contains comparably high percentages of cells expressing transcription factors critical for LSC stemness maintenance (p63) and corneal epithelial differentiation (PAX6). Preclinical studies confirmed local engraftment potential of the cells and gave no signals of toxicity and tumorgenicity. These findings were sufficient for the product to be approved by the German Paul Ehrlich Institute and the U.S. Food & Drug Administration to be tested in an international multicenter phase I/IIa clinical trial (NCT03549299) to evaluate the safety and therapeutic efficacy in patients with LSCD.ConclusionBuilding upon these data in conjunction with the previously shown cornea-restoring capacity of human ABCB5+ LSCs in animal models of LSCD, we provide an advanced allogeneic LSC-based treatment strategy that shows promise for replenishment of the patient’s LSC pool, recreation of a functional barrier against invading conjunctival cells and restoration of a transparent, avascular cornea.

Comparison of the Migration Potential through Microperforated Membranes of CD146+ GMSC Population versus Heterogeneous GMSC Population.

Background Guided tissue regeneration (GTR) is a powerful modality for periodontal regeneration, but it blocks the periosteum and gingival stem cells (GMSCs), from supporting periodontal wound by the nutrients, growth factors, and regenerative cells. The microperforated membrane considered a rewarding solution for this major drawback; GMSCs can migrate through a GTR microperforated membrane toward a chemoattractant, with the blocking of other unfavorable epithelial cells and fibroblasts. In the absence of a sole marker for MSC, a homogeneous population of GMSC is difficult to isolate; using CD146 as confirmatory markers for MSC identification, testing the behaviour of such homogeneous population in migration dynamics was the question to answer in this study. Materials and Methods GMSCs from healthy crown lengthening tissue was isolated (n = 3), its stem cell nature was confirmed, CD146 and CD271 markers were confirmatory markers to confirm homogenous stem cell population, and magnetic sorting was used to isolate GMSC with CD146 markers. A homogenous CD146 population was compared to heterogeneous GMSCs of origin; the population doubling time and MTT test of the two populations were compared. Migration dynamics were examined in a transwell migration chamber through 8 μm perforated polycarbonic acid membrane, and 0.4 μm and 3 μm perforated collagen-coated polytetrafluoroethylene membrane (PTFE) and 10% fetal bovine serum (FBS) were the chemoattractants used in the lower compartment to induce cell migration, were incubated in a humidified environment for 24 hours, then migrated the cell in the lower compartment examined by a light and electron microscope. Results GMSCs fulfilled all the minimal criteria of stem cells and showed low signal 10% for CD146 on average and extremely low signal 2% for CD271 on average. Magnetic sorting optimized the signal of CD146 marker to 55%. GMSC CD146 population showed nonstatistically significant shorter population doubling time. CD146 homogeneous population migrated cell numbers were statistically significant compared to the heterogeneous population, through 0.4 μm and 3 μm perforated collagen membrane and 8 μm perforated polycarbonate membrane. Scanning electron microscopy proved the migration of the cells. Conclusions A subset of the isolated GMSC showed a CD146 marker, which is considered a dependable confirmatory marker for the stem cells. In terms of GMSC migration through the microperforated membrane, a homogeneous CD146 population migrates more statistically significant than a heterogeneous GMSC population.

Sequencing-Based Protein Analysis of Single Extracellular Vesicles.

Circulating extracellular vesicles (EVs)-biological nanomaterials shed from most mammalian cells-have emerged as promising biomarkers, drug delivery vesicles, and treatment modulators. While different types of vesicles are being explored for these applications, it is becoming clear that human EVs are quite heterogeneous even in homogeneous or monoclonal cell populations. Since it is the surface EV protein composition that will largely dictate their biological behavior, high-throughput single EV profiling methods are needed to better define EV subpopulations. Here, we present an antibody-based immunosequencing method that allows multiplexed measurement of protein molecules from individual nanometer-sized EVs. We use droplet microfluidics to compartmentalize and barcode individual EVs. The barcodes/antibody-DNA are then sequenced to determine protein composition. Using this highly sensitive technology, we detected specific proteins at the single EV level. We expect that this technology can be further adapted for multiplexed protein analysis of any nanoparticle.

Comparison of immune modulatory properties of human multipotent mesenchymal stromal cells derived from bone marrow and placenta

ABSTRACT Multipotent mesenchymal stromal cells (MSC) can be isolated from many tissues, including bone marrow (BM) and placenta (PL). Human placenta can be obtained readily without invasive procedures. There may be differences, however, in differentiation capacity and immunomodulation by MSC isolated from BM or PL. The early pregnancy factor (heat shock protein 10; EPF/Hsp10) is a small protein that exhibits immunomodulatory properties. We compared BM- and PL-MSC, and assessed their efficacy for suppressing T-cell proliferation in vitro and the role of EPF/Hsp10 in this process. PL-MSC were collected from whole placenta after removal of the amniotic and chorionic membranes followed by serial enzymatic digestions. The PL-MSC were compared to BM-MSC, obtained from healthy donors. Differentiation capacity, cytokine secretion, expression and secretion of immunomodulatory molecules, immunophenotype and real time proliferation were assessed using cytokine arrays, ELISA assays, flow cytometry, immunohistochemical staining and western blotting. Whereas BM-MSC consisted of a homogeneous cell population with strong expression of mesenchymal markers, PL-MSC consisted of a mixed population of cells with variable CD73, CD90 and CD105 expression. PL-MSC exhibited a significantly greater proliferation rate than BM-MSC. The presence of both stem cells and more mature cells in the PL-MSC cultures resulted in decreased differentiation capacity and reduced efficacy of immune suppression in co-cultures with T-cells. Although robust intracellular expression of EPF/Hsp10 in both BM- and PL-MSC was observed, secretion of the protein in response to immune activating stimuli remained below detectable levels. Secretion of pro-inflammatory cytokines was significantly greater in BM-MSC than PL-MSC, whereas no difference was observed in the secretion of hematopoiesis supporting growth factors. Development of culture methods for isolation of pure populations of PL-MSC may improve the quality of the product and reproducibility of results.

Migratory cortical interneuron-specific transcriptome abnormalities in schizophrenia

Cortical interneurons (cINs) are substantially affected in Schizophrenia (SCZ) and enriched for SCZ heritability during development. To understand SCZ-specific changes in these cells during development, we isolated migratory cINs from cIN spheres derived from 5 healthy control (HC) and 5 SCZ induced pluripotent stem cell lines (iPSCs). Transcriptome analyses show dysregulation in extracellular matrix pathways as the major disturbances in SCZ migratory cINs, whereas sphere cINs show dysregulation in immune pathways. This result suggests the importance of using homogeneous cell populations to identify stage-specific abnormalities and provides a platform to further study the biology of schizophrenia pathogenesis during early development.

Chimeric Antigen Receptor T Cells as Senolytic and Antifibrotic Therapy.

During chronic liver injury, hepatic stellate cells (HSC) differentiate from lipid-storing pericytes into extracellular matrix (ECM)-producing fibroblasts. While we tend to view these activated HSC as a homogenous fibrogenic cell population that accumulates in the fibrotic liver, this oversimplifies the dynamic nature of fibrogenesis, the different functional states of HSC and their ultimate fate. In parallel to their activation, HSC also undergo massive expansion under stimulation of cytokines such as platelet-derived growth factor beta, leading to their accumulation within fibrotic septa and ultimately HSC senescence. In a recent paper published in Nature, Amor et al. establish chimeric antigen receptor (CAR) T cells as new and powerful method for clearing senescent-cells and demonstrate that removal of senescent HSC ameliorates liver fibrosis (1).

Dielectrophoretic Characterization of Tenogenically Differentiating Mesenchymal Stem Cells.

Tendons are collagenous musculoskeletal tissues that connect muscles to bones and transfer the forces necessary for movement. Tendons are susceptible to injury and heal poorly, with long-term loss of function. Mesenchymal stem cell (MSC)-based therapies are a promising approach for treating tendon injuries but are challenged by the difficulties of controlling stem cell fate and of generating homogenous populations of stem cells optimized for tenogenesis (differentiation toward tendon). To address this issue, we aim to explore methods that can be used to identify and ultimately separate tenogenically differentiated MSCs from non-tenogenically differentiated MSCs. In this study, baseline and tenogenically differentiating murine MSCs were characterized for dielectric properties (conductivity and permittivity) of their outer membrane and cytoplasm using a dielectrophoretic (DEP) crossover technique. Experimental results showed that unique dielectric properties distinguished tenogenically differentiating MSCs from controls after three days of tenogenic induction. A single shell model was used to quantify the dielectric properties and determine membrane and cytoplasm conductivity and permittivity. Together, cell responses at the crossover frequency, cell morphology, and shell models showed that changes potentially indicative of early tenogenesis could be detected in the dielectric properties of MSCs as early as three days into differentiation. Differences in dielectric properties with tenogenesis indicate that the DEP-based label-free separation of tenogenically differentiating cells is possible and avoids the complications of current label-dependent flow cytometry-based separation techniques. Overall, this work illustrates the potential of DEP to generate homogeneous populations of differentiated stem cells for applications in tissue engineering and regenerative medicine.

Segregation of brain and organizer precursors is differentially regulated by Nodal signaling at blastula stage.

ABSTRACTThe blastula Chordin- and Noggin-expressing (BCNE) center comprises animal-dorsal and marginal-dorsal cells of the amphibian blastula and contains the precursors of the brain and the gastrula organizer. Previous findings suggested that the BCNE behaves as a homogeneous cell population that only depends on nuclear β-catenin activity but does not require Nodal and later segregates into its descendants during gastrulation. In contrast to previous findings, in this work, we show that the BCNE does not behave as a homogeneous cell population in response to Nodal antagonists. In fact, we found that chordin.1 expression in a marginal subpopulation of notochordal precursors indeed requires Nodal input. We also establish that an animal BCNE subpopulation of cells that express both, chordin.1 and sox2 (a marker of pluripotent neuroectodermal cells), and gives rise to most of the brain, persisted at blastula stage after blocking Nodal. Therefore, Nodal signaling is required to define a population of chordin.1+ cells and to restrict the recruitment of brain precursors within the BCNE as early as at blastula stage. We discuss our findings in Xenopus in comparison to other vertebrate models, uncovering similitudes in early brain induction and delimitation through Nodal signaling.This article has an associated First Person interview with the first author of the paper.

Pluripotent stem cell-derived epithelium misidentified as brain microvascular endothelium requires ETS factors to acquire vascular fate

Cells derived from pluripotent sources in vitro must resemble those found in vivo as closely as possible at both transcriptional and functional levels in order to be a useful tool for studying diseases and developing therapeutics. Recently, differentiation of human pluripotent stem cells (hPSCs) into brain microvascular endothelial cells (ECs) with blood-brain barrier (BBB)-like properties has been reported. These cells have since been used as a robust in vitro BBB model for drug delivery and mechanistic understanding of neurological diseases. However, the precise cellular identity of these induced brain microvascular endothelial cells (iBMECs) has not been well described. Employing a comprehensive transcriptomic metaanalysis of previously published hPSC-derived cells validated by physiological assays, we demonstrate that iBMECs lack functional attributes of ECs since they are deficient in vascular lineage genes while expressing clusters of genes related to the neuroectodermal epithelial lineage (Epi-iBMEC). Overexpression of key endothelial ETS transcription factors (ETV2, ERG, and FLI1) reprograms Epi-iBMECs into authentic endothelial cells that are congruent with bona fide endothelium at both transcriptomic as well as some functional levels. This approach could eventually be used to develop a robust human BBB model in vitro that resembles the human brain EC in vivo for functional studies and drug discovery.