Cell Doublet Research Articles

The scaffold protein IQGAP1 promotes reorientation of epithelial cell polarity at the two-cell stage for cystogenesis.

A single epithelial cell embedded in extracellular matrix (ECM) can proliferate to form an apical lumen-harboring cyst, whose formation is a fundamental step in epithelial organ development. At an early two-cell stage after cell division, the cell doublet typically displays "inverted" polarity, with apical and basolateral proteins being located to the ECM-facing and cell-cell-contacting plasma membranes, respectively. Correct cystogenesis requires polarity reorientation, a process containing apical protein endocytosis from the ECM-abutting periphery and subsequent apical vesicle delivery to a cell-cell contact site for lumen formation. Here, we show that downstream of the ECM-signal-transducer β1-integrin, Rac1, and its effector IQGAP1 promote apical protein endocytosis, contributing to polarity reorientation of mammalian epithelial Madin-Darby canine kidney (MDCK) cells at a later two-cell stage in three-dimensional culture. Rac1-GTP facilitates IQGAP1 interaction with the Rac-specific activator Tiam1, which also contributes to the endocytosis and enhances the effect of IQGAP1. These findings suggest that Tiam1 and IQGAP1 form a positive feedback loop to activate Rac1. With Rac1-GTP, IQGAP1 also binds to AP2α, an adaptor protein subunit for clathrin-mediated endocytosis; depletion of the AP2 complex impairs apical protein endocytosis in MDCK doublets. Thus, Rac1 likely participates in polarity reorientation at the two-cell stage via its interaction with IQGAP1.

Polarity-driven three-dimensional spontaneous rotation of a cell doublet

Mechanical interactions between cells play a fundamental role in the self-organization of organisms. How these interactions drive coordinated cell movement in three dimensions remains unclear. Here we report that cell doublets embedded in a three-dimensional extracellular matrix undergo spontaneous rotations. We investigate the rotation mechanism and find that it is driven by a polarized distribution of myosin within cell cortices. The mismatched orientation of this polarized distribution breaks the doublet mirror symmetry. In addition, cells adhere at their interface through adherens junctions and with the extracellular matrix through focal contacts near myosin clusters. We use a physical theory describing the doublet as two interacting active surfaces to show that rotation is driven by myosin-generated gradients of active tension whose profiles are dictated by interacting cell polarity axes. We also show that three-dimensional shape symmetries are related to broken symmetries of the myosin distribution in cortices. To test for the rotation mechanism, we suppress myosin clusters using laser ablation and generate new myosin clusters by optogenetics. Our work clarifies how polarity-oriented active mechanical forces drive collective cell motion in three dimensions.

Force propagation between epithelial cells depends on active coupling and mechano-structural polarization.

Cell-generated forces play a major role in coordinating the large-scale behavior of cell assemblies, in particular during development, wound healing, and cancer. Mechanical signals propagate faster than biochemical signals, but can have similar effects, especially in epithelial tissues with strong cell-cell adhesion. However, a quantitative description of the transmission chain from force generation in a sender cell, force propagation across cell-cell boundaries, and the concomitant response of receiver cells is missing. For a quantitative analysis of this important situation, here we propose a minimal model system of two epithelial cells on an H-pattern ('cell doublet'). After optogenetically activating RhoA, a major regulator of cell contractility, in the sender cell, we measure the mechanical response of the receiver cell by traction force and monolayer stress microscopies. In general, we find that the receiver cells show an active response so that the cell doublet forms a coherent unit. However, force propagation and response of the receiver cell also strongly depend on the mechano-structural polarization in the cell assembly, which is controlled by cell-matrix adhesion to the adhesive micropattern. We find that the response of the receiver cell is stronger when the mechano-structural polarization axis is oriented perpendicular to the direction of force propagation, reminiscent of the Poisson effect in passive materials. We finally show that the same effects are at work in small tissues. Our work demonstrates that cellular organization and active mechanical response of a tissue are key to maintain signal strength and lead to the emergence of elasticity, which means that signals are not dissipated like in a viscous system, but can propagate over large distances.

Atlas-scale single-cell chromatin accessibility using nanowell-based combinatorial indexing.

Here we present advancements in single-cell combinatorial indexed ATAC-seq (sciATAC) to measure chromatin accessibility that leverage nanowell chips to achieve atlas-scale cell throughput (>105) at low cost. The platform leverages the core of the sciATAC workflow where multiple indexed tagmentation reactions are performed, followed by pooling and distribution to a second set of reaction wells for PCR-based indexing. In this work, we instead leverage a chip containing 5,184 nanowells as the PCR stage of indexing, enabling a 52-fold improvement in scale and reduction in per-cell preparation costs. We detail three variants that balance cell throughput and depth of coverage and apply these methods to banked mouse brain tissue, producing maps of cell types as well as neuronal subtypes that includes integration with exiting scATAC and scRNA-seq datasets. Our optimized workflow achieves a high fraction of reads that fall within called peaks (>80%) and low cell doublet rates. The high cell coverage technique produces high unique reads per cell, while retaining high enrichment for open chromatin regions, enabling the assessment of >70,000 unique accessible on average for each cell profiled. When compared to current methods in the field, our technique provides similar or superior per-cell information with very low levels of cell-to-cell crosstalk, and achieves this at a cost point much lower than existing assays.

Interacting active surfaces: A model for three-dimensional cell aggregates.

We introduce a modelling and simulation framework for cell aggregates in three dimensions based on interacting active surfaces. Cell mechanics is captured by a physical description of the acto-myosin cortex that includes cortical flows, viscous forces, active tensions, and bending moments. Cells interact with each other via short-range forces capturing the effect of adhesion molecules. We discretise the model equations using a finite element method, and provide a parallel implementation in C++. We discuss examples of application of this framework to small and medium-sized aggregates: we consider the shape and dynamics of a cell doublet, a planar cell sheet, and a growing cell aggregate. This framework opens the door to the systematic exploration of the cell to tissue-scale mechanics of cell aggregates, which plays a key role in the morphogenesis of embryos and organoids.

Accurate identification of cell doublet profiles: Comparison of light scattering with fluorescence measurement techniques.

Doublet discrimination is usually based on pulse analysis of light scatter parameters. A combination of two pulse parameters (Area, A; Height, H; or Width, W) can be used to discriminate a pulse originated in a single cell from a pulse originated from cells stuck together. Fluorescence signals can be also used to discriminate aggregates, being essential to identify cells in the G2/M phase from doublets in the G0/G1 phase in cell cycle/DNA applications. The most used method combines FSC-A versus FSC-H, whereas other strategies combine FSC-H versus FSC-W, SSC-H versus SSC-A and SSC-H versus SSC-W. However, when studying activated or proliferating cells, scatter discrimination can be difficult. In this study, we have compared the use of light scattering with fluorescence measurement techniques for successful doublet discrimination for single cells. Effective use of FSC and SSC height, area and width are commonly used to eliminate aggregates. However, fluorescence-based methods using viable DNA stains provide a good compromise between performance and accurate manual gating methods, especially for highly concentrated cell products and pathological specimens. Viable DNA dyes, such as Vybrant™ DyeCycle™ Violet stain or Hoechst 33342, can be used to detect nucleated cells in blood and in bone marrow, or to discriminate cell aggregates and debris based on no-lyse no-wash assays, where scatter degradation is a dominant component of the measured data, which increases with event rate.

Cortical tension initiates the positive feedback loop between cadherin and F-actin

Adherens junctions physically link two cells at their contact interface via extracellular binding between cadherin molecules and intracellular interactions between cadherins and the actin cytoskeleton. Cadherin and actomyosin cytoskeletal dynamics are regulated reciprocally by mechanical and chemical signals, which subsequently determine the strength of cell-cell adhesions and the emergent organization and stiffness of the tissues they form. However, an understanding of the integrated system is lacking. We present a new mechanistic computational model of intercellular junction maturation in a cell doublet to investigate the mechanochemical cross talk that regulates adherens junction formation and homeostasis. The model couples a two-dimensional lattice-based simulation of cadherin dynamics with a reaction-diffusion representation of the reorganising actomyosin network through its regulation by Rho signalling at the intracellular junction. We demonstrate that local immobilization of cadherin induces cluster formation in a cis-less-dependent manner. We then recapitulate the process of cell-cell contact formation. Our model suggests that cortical tension applied on the contact rim can explain the ring distribution of cadherin and actin filaments (F-actin) on the cell-cell contact of the cell doublet. Furthermore, we propose and test the hypothesis that cadherin and F-actin interact like a positive feedback loop, which is necessary for formation of the ring structure. Different patterns of cadherin distribution were observed as an emergent property of disturbances of this positive feedback loop. We discuss these findings in light of available experimental observations on underlying mechanisms related to cadherin/F-actin binding and the mechanical environment.

Morphodynamic signatures of MDA-MB-231 single cells and cell doublets undergoing invasion in confined microenvironments

Cancer cell metastasis is a major factor in cancer-related mortality. During the process of metastasis, cancer cells exhibit migratory phenotypes and invade through pores in the dense extracellular matrix. However, the characterization of morphological and subcellular features of cells in similar migratory phenotypes and the effects of geometric confinement on cell morphodynamics are not well understood. Here, we investigate the phenotypes of highly aggressive MDA-MB-231 cells in single cell and cell doublet (an initial and simplified collective state) forms in confined microenvironments. We group phenotypically similar single cells and cell doublets and characterize related morphological and subcellular features. We further detect two distinct migratory phenotypes, fluctuating and non-fluctuating, within the fast migrating single cell group. In addition, we demonstrate an increase in the number of protrusions formed at the leading edge of cells after invasion through geometric confinement. Finally, we track the short and long term effects of varied degrees of confinement on protrusion formation. Overall, our findings elucidate the underlying morphological and subcellular features associated with different single cell and cell doublet phenotypes and the impact of invasion through confined geometry on cell behavior.

Deconvolution of Donor and Recipient Cells from Lung Transplant Single Cell RNA-seq Data

<h3>Purpose</h3> Single-cell RNA-sequencing (scRNA-seq) is a powerful tool to reveal novel insights into transplant biology but a key requirement is the robust identification of donor and recipient cells. Expressed single nucleotide variants (SNVs) can be used to differentiate these cells; however, drastic differences in donor and recipient cell proportions and cell types challenge current methods. We developed "scTx", which mitigates these issues and reliably dentifies donor and recipient cells without matched genotype data. <h3>Methods</h3> scTX, has two steps: 1) identification of donor and recipient genotypes using expressed SNVs, and 2) assignment of each cell to a genotype or cell doublet. We validate our algorithm using simulated lung transplant reperfusion data by mixing scRNA-seq lung data with peripheral blood mononuclear cell data at different ratios and amounts of ambient RNA. We apply our method to four post-transplant bronchoalveolar lavage (BAL) samples and two explanted lungs with chronic lung allograft dysfunction (CLAD). <h3>Results</h3> Using simulated data, scTX can accurately identify donor and recipient cells at low proportions (i.e. < 1% of the total cells) and does not identify a second genotype when one is not present. We show that scTx reliably identifies two genotypes from CLAD and BAL samples and that their donor and recipient origin can be inferred based on annotated cell types (Fig 1). <h3>Conclusion</h3> scTx can reliably deconvolute donor from recipient cells in lung transplant samples with mixed cell types and proportions. This broadly applicable method will unravel mechanisms of rejection and reveal the dynamics of donor and recipient cell populations in human datasets.

MUC16 suppresses human and murine innate immune responses

ObjectiveMUC16, the mucin that contains the CA125 epitopes, suppresses the cytolytic responses of human NK cells and inhibits the efficacy of therapeutic antibodies. Here, we provide further evidence of the regulatory role of MUC16 on human and murine NK cells and macrophages. MethodsTarget cell cytolysis and doublet formation assays were performed to assess effects of MUC16 on human NK cells. The effect of MUC16 on ovarian tumor growth was determined in a mouse model by monitoring survival and ascites formation. Innate immune cells from spleens and peritoneal cavities of mice were isolated and stimulated in vitro with anti-CD40 antibody, lipopolysaccharide and IFN-γ and their ability to cytolyse MUC16 expressing and non-expressing cells was determined. ResultsWe confirm that MUC16 inhibits cytolysis by human NK cells as well as the formation of NK-tumor conjugates. Mice implanted with MUC16-knockdown OVCAR-3 show >2-fold increase in survival compared to controls. Murine NK cells and macrophages are more efficient at lysing MUC16-knockdown cells. In vitro cytotoxicity assays with NK cells and macrophages isolated from mice stimulated with anti-CD40 antibody showed 2–3-fold increased activity against the MUC16-knockdown cells as compared to matching target cells expressing this mucin. Finally, knockdown of MUC16 increased the susceptibility of cancer cells to ADCC by murine splenocytes. ConclusionsFor the first time, we demonstrate the immunoregulatory effects of MUC16 on murine NK cells and macrophages. Our study implies that the immunoregulatory role of MUC16 on murine NK cells and macrophages should be considered when examining the biology of MUC16 in mouse models.

Barcoding of live human peripheral blood mononuclear cells for multiplexed mass cytometry.

Mass cytometry is developing as a means of multiparametric single-cell analysis. In this study, we present an approach to barcoding separate live human PBMC samples for combined preparation and acquisition on a cytometry by time of flight instrument. Using six different anti-CD45 Ab conjugates labeled with Pd104, Pd106, Pd108, Pd110, In113, and In115, respectively, we barcoded up to 20 samples with unique combinations of exactly three different CD45 Ab tags. Cell events carrying more than or less than three different tags were excluded from analyses during Boolean data deconvolution, allowing for precise sample assignment and the electronic removal of cell aggregates. Data from barcoded samples matched data from corresponding individually stained and acquired samples, at cell event recoveries similar to individual sample analyses. The approach greatly reduced technical noise and minimizes unwanted cell doublet events in mass cytometry data, and it reduces wet work and Ab consumption. It also eliminates sample-to-sample carryover and the requirement of instrument cleaning between samples, thereby effectively reducing overall instrument runtime. Hence, CD45 barcoding facilitates accuracy of mass cytometric immunophenotyping studies, thus supporting biomarker discovery efforts, and it should be applicable to fluorescence flow cytometry as well.

Actin dynamics modulate mechanosensitive immobilization of E-cadherin at adherens junctions.

Mechanical stress is increasingly being shown to be a potent modulator of cell-cell junctional morphologies in developmental and homeostatic processes. Intercellular force sensing is thus expected to be an important regulator of cell signalling and tissue integrity. In particular, the interplay between myosin contractility, actin dynamics and E-cadherin recruitment largely remains to be uncovered. We devised a suspended cell doublet assay to quantitatively assess the correlation between myosin II activity and local E-cadherin recruitment. The single junction of the doublet exhibited a stereotypical morphology, with E-cadherin accumulating into clusters of varied concentrations at the rim of the circular contact. This local recruitment into clusters derived from the sequestration of E-cadherin through a myosin-II-driven modulation of actin turnover. We exemplify how the regulation of actin dynamics provides a mechanism for the mechanosensitive response of cell contacts.

Real-Time Quantitative Measurement of Myosin and Strain Dependent Recruitment of E-Cad in Spot Adherens Junctions

The response of adherens junction to mechanical stress is a key process in cell-cell contact reorganization that remains poorly understood partly owing to the lack of appropriate measurement techniques. In this paper, we present an innovative micro-well based approach to image at high resolution the horizontal adherens junction of a suspended vertical cell doublet during contact elongation and under mechanical constrain. We find that the E-cadherins cluster in submicron sized puncta at the edge the actin depleted contact area. They form an almost periodic ring-like structure that displays spatially organized fluctuations of E-cad recruitment levels. We demonstrate by real time imaging that these fluctuations correlate with the local contact deformations induced by local myosin contractility gradients. Finally we establish a unified relationship between the local strain and the changes in E-cad recruitment levels induced by global contractility changes, local gradients and external mechanical stimuli. We hence provide the first quantitative real time measurement of the response of an adherens junction to mechanical strain. We propose the existence of an adaptive pathway with a fast and strong response for weak mechanical stimuli.View Large Image | View Hi-Res Image | Download PowerPoint Slide

A microdevice for studying intercellular electromechanical transduction in adult cardiac myocytes

Intercellular electromechanical transduction in adult cardiac myocytes plays an important role in regulating heart function. The efficiency of intercellular electromechanical transduction has so far been investigated only to a limited extent, which is largely due to the lack of appropriate tools that can quantitatively assess the contractile performance of interconnected adult cardiac myocytes. In this paper we report a microengineered device that is capable of applying electrical stimulation to the selected adult cardiac myocyte in a longitudinally connected cell doublet and quantifying the intercellular electromechanical transduction by measuring the contractile performance of stimulated and un-stimulated cells in the same doublet. The capability of applying selective electrical stimulation on only one cell in the doublet is validated by examining cell contractile performance while blocking the intercellular communication. Quantitative assessment of cell contractile performance in isolated adult cardiac myocytes is performed by measuring the change in cell length. The proof-of-concept assessment of gap junction performance shows that the device is useful in studying the efficiency of gap junctions in adult cardiac myocytes, which is most relevant to the synchronized pumping performance of native myocardium. Collectively, this work provides a quantitative tool for studying intercellular electromechanical transduction and is expected to develop a comprehensive understanding of the role of intercellular communication in various heart diseases.

Evaluation of intermolecular forces in a circulating system

Intercellular interactions, which are mediated by a variety of complex intercellular molecules through the processes of formation and dissociation of molecular bonds, play a critical role in regulating cellular functions in biological systems. Various approaches are applied to evaluate intercellular or molecular bonding forces. To quantify the intermolecular interaction forces, flow chamber has become a meaningful technique as it can ultimately mimic the cellular microenvironment in vivo under physiological flow conditions. Hydrodynamic forces are usually used to predict the intercellular forces down to the single molecular level. However, results show that only using hydrodynamic force will overestimate up to 30% of the receptor–ligand strength when the non-specific forces such as Derjaguin–Landau–Verway–Overbeek (DLVO) forces become un-neglected. Due to the nature of high ion concentration in the physiological condition, electrostatic force is largely screened which will cause DLVO force unbalanced. In this study, we propose to take account of the DLVO force, including van der Waals (VDW) force and electrostatic force, to predict the intermolecular forces of a cell doublet and cell–substrate model in a circulating system. Results also show that the DLVO force has a nonlinear effect as the cell–cell or cell–substrate distance changes. In addition, we used the framework of high accuracy hydrodynamic theories proved in colloidal systems. It is concluded that DLVO force could not be ignored in quantitative studies of molecular interaction forces in circulating system. More accurate prediction of intercellular forces needs to take account of both hydrodynamic force and DLVO force.

Obligatory Asymmetric Cell Division Regulates Self-Renewal In Hematopoietic Progenitor/Stem Cells

AbstractAbstract 571Asymmetric cell division, a proposed mechanism by which hematopoietic progenitor/stem cells (HPSC) maintain a balance between self-renewal and differentiation, has rarely been observed. Here we report the surprising finding that cultured mouse primary HPSC routinely generate pairs of daughter cells with 2 distinct phenotypes after a single round of cell division. Mouse bone marrow cells were cultured on chamber slides in the presence of stem cell factor (SCF). BrdU was added overnight to label dividing cells, and the cells were examined by immunofluorescence microscopy on day 2–4 of culture. In each BrdU+c-Kit+ divided cell doublet, c-Kit was invariably expressed in only 1 of the 2 daughter cells. In contrast, the other daughter cell was negative for c-Kit but positive for the asymmetric cell fate determinant Numb and mature myeloid markers Mac1, Gr1, M-CSFR and F4/80. Similarly, in each BrdU+Sca1+ cell doublet, 1 daughter cell was positive for the stem cell markers Sca1, c-Kit, CD150 and CD201, whereas the other cell was negative for these markers but positive for Numb and the mature myeloid markers. Analysis of 400 such doublets showed that the probability of HPSC undergoing asymmetric division was 99.5% (95% confidence interval 98–100%), indicating that asymmetric division in HPSC is in fact not rare but obligatory. In other model systems, it has been shown that activation of the atypical protein kinase C (aPKC)-Par6-Par3 cell polarity complex and realignment of the microtubule cytoskeleton precede asymmetric cell division. We asked whether similar steps are involved in the asymmetric division of HPSC. We found that c-Kit receptors, upon stimulation by SCF, rapidly capped at an apical pole next to the microtubule-organizing center, followed by redistribution to the same pole of the aPKC-Par6-Par3 complex and microtubule-stabilizing proteins APC, β-catenin, EB1 and IQGAP1. Strikingly, after cell division, the aPKC-Par6-Par3 complex and other polarity markers all partitioned only into the c-Kit+/Sca1+ daughter cell and not the mature daughter cell. The acetylated and detyrosinated forms of stabilized microtubules were also present only in the c-Kit+/Sca1+ cell, as were the Aurora A and Polo-like kinases, 2 mitotic kinases associated with asymmetric cell division. To understand how c-Kit activation triggers downstream polarization events, we studied the role of lipid rafts, cholesterol-enriched microdomains in the cell membrane that serve as organization centers of signaling complexes. These are enriched in phosphatidylinositol 4,5-bisphosphate and annexin 2, putative attachment sites for the aPKC-Par6-Par3 complex. We found that SCF stimulation led to coalescence of lipid raft components at the site of the c-Kit cap, and treatment with a wide range of inhibitors that blocked lipid raft formation abrogated polarization of the aPKC-Par6-Par3 complex and division of the c-Kit+/Sca1+ cells. Because obligatory asymmetric division in cultured HPSC would prevent a net increase in their number, we sought a way to bypass its mechanism. We tested whether inhibition of protein phosphatase 2A (PP2A), a physiological antagonist of aPKC, would enhance aPKC activity and promote self-renewal of HPSC. Treatment of cultured HPSC with okadaic acid or calyculin, 2 well-characterized PP2A inhibitors, increased the percent of c-Kit+/Sca1+ cells undergoing symmetric division from 0% to 23.3% (p<0.001). In addition, small colonies comprised of symmetrically dividing cells uniformly positive for Sca1, c-Kit, CD150 and CD201 were noted in the culture. To functionally characterize the effect of PP2A inhibition, mouse bone marrow cells were cultured in the absence or presence of PP2A inhibitors and transplanted into irradiated congenic mice in a competitive repopulation assay. At 4–8 weeks post-transplant, the donor engraftment rate increased from ∼1 in mice transplanted with untreated cells to >30% in mice transplanted with PP2A inhibitor-treated cells. This dramatic increase indicates that PP2A inhibition can effectively perturb the mechanism of asymmetric cell division and promote the self-renewal of HPSC. In summary, our data showed that obligatory asymmetric cell division works to maintain a strict balance between self-renewal and differentiation in HPSC and pharmacological manipulation of the cell polarity machinery could potentially be used to expand HPSC for clinical use. Disclosures:No relevant conflicts of interest to declare.

Symmetry-breaking Induction in Cleavage-Arrested Cell Doublet from Ascidian Two Cell Embryo Produce an Organizer Model

Symmetry-breaking Induction in Cleavage-Arrested Cell Doublet from Ascidian Two Cell Embryo Produce an Organizer Model

Sporogony of Tetracapsuloides bryosalmonae in the brown trout Salmo trutta and the role of the tertiary cell during the vertebrate phase of myxozoan life cycles

Tetracapsuloides bryosalmonae is the myxozoan that causes the commercially and ecologically important proliferative kidney disease of salmonid fish species. Immunohistochemistry and electron microscopy were used to examine the development of this parasite within the kidney of the brown trout Salmo trutta. The main replicative phase of T. bryosalmonae is a cell doublet composed of a primary cell and a single secondary cell. Engulfment of one secondary cell by another to form a secondary-tertiary doublet (S-T doublet) heralded the onset of sporogony whereupon the parasite migrated to the kidney tubule lumen. Within the tubule, the parasite transformed into a pseudoplasmodium and anchored to the tubule epithelial cells via pseudopodial extensions. Within each pseudoplasmodium developed a single spore, composed of 4 valve cells, 2 polar capsules and 1 sporoplasm. The pseudoplasmodia formed clusters suggesting that large numbers of spores develop within the fish. This examination of T. bryosalmonae suggests that the main replicative phase of freshwater myxozoans within vertebrates is via direct replication of cell doublets rather than through the rupturing of extrasporogonic stages, while tertiary cell formation relates only to sporogony. Taken in conjunction with existing phylogenetic data, 5 distinct sporogonial sequences are identified for the Myxozoa.

Morphology of small aggregates of red blood cells

Blood can be considered a two-phase liquid composed of plasma as well as cells and cell aggregates. The degree of cell aggregation is an important determinant of blood rheology: The size and shape of the aggregates affect blood viscosity. The microscopic mechanisms of red blood cell adhesion involve a complex interplay of electrostatic, van der Waals, and a range of specific biochemical inter-membrane interactions. Here we use an effective model of these interactions combined with the membrane elasticity theory to calculate the equilibrium shape of a red blood cell doublet and compare it with the experimentally observed red blood cell aggregates both in vitro and in vivo. Special attention is devoted to the shape of doublets formed by dissimilar cells. A possible effect of doublet shape on pathways of the formation of multicellular aggregates is discussed. Red blood cell rouleau formation is expected to take place at intermediate adhesion strengths where the outer doublet surfaces are either concave or flat, whereas in the strong-adhesion regime where the outer doublet surfaces are convex the cells should form rounded clump-like aggregates.

Identification of individual CD34+lin− daughter cells that retain multilineage differentiation potential after one division

There is a need to determine whether culture conditions may exist which favor solely proliferative self-renewal of hematopoietic stem cells as opposed to proliferation with differentiation. A key to answering this is the ability to detect self-renewal in vitro. We have previously reported that TPO and FLT-3L can interact to promote doubling and phenotypic self-renewal of single cord blood CD34+Thy 1+lin− (lin−) cells and this cell fate/phenotype decision can be detected s early as the first cell division. To determine if individual daughter cells that remained lin− after 1 division still retained lymphoid/myeloid differentiation potential, single cell micromanipulation of doublets was performed. CB lin− cells were plated in Terasaki plates containing FLT3L+TPO. Each well was monitored daily for evidence of cell division. When a division was detected the cell doublet was stained for phenotype assessment and daughter cells that remained lin− were separated by micromanipulation and placed in different wells on AFT024 stroma with IL-7, SCF, FLT-3L. After 28 days, wells in which the single cell had proliferated were stained with FITC and PE-conjugated antibodies against lymphoid (CD2/3, CD16, CD19, CD56) and myeloid (CD13, CD15, CD33) specific markers to identify which cells of the doublet had multi-lineage differentiation potential. In preliminary experiments, with 783 single cells, 52% of the cells that divided, over 7 days, remained lin− (405 doublets). Of the 810 total cells, 73.5% of those were successfully separated and transferred to stroma. Staining, revealed that 27 doublets (90%) had at least 1 daughter cell that generated progeny with both L/M markers, and that 23% of the doublets separated had both daughters capable of generating L/M positive progeny. This assay may be useful to improve in vitro stem cell expansion conditions by identifying which single cells with retained phenotype can generate progeny with multilineage potential.