Single Cell Deposition Research Articles

Endothelial Cells Derived from Human Vessel Walls Contain a Complete Hierarchy of Endothelial Progenitor Cells.

Endothelial progenitor cells (EPCs) can be isolated from adult peripheral and umbilical cord blood. EPCs are thought to originate from bone marrow, circulate in peripheral blood, and migrate to sites of angiogenesis. However, the number of circulating EPCs in peripheral blood is remarkably low, and recent genetic studies show that the contribution of bone marrow derived EPCs to newly formed vascular networks is minimal. Further, while endothelial cells (ECs) derived from vessel walls are widely considered to be differentiated mature ECs, these cells retain extensive proliferative potential and can be passaged for at least 40 population doubling in vitro. Based on these observations, we tested whether EPCs potentially reside in vessel walls utilizing a newly developed single cell deposition assay (Blood, 2004). Analogous to a paradigm established in the hematopoietic cell system, we can utilize this assay to reproducibly identify the following EPCs: (1) high proliferative potential - endothelial colony forming cells (HPP-ECFC), which form macroscopic colonies that form secondary and tertiary colonies upon replating, (2) low proliferative potential - endothelial colony forming cells (LPP-ECFC), which form colonies greater than 50 cells, but do not form secondary colonies upon replating, (3) endothelial cell clusters (EC-clusters) that contain less than 50 cells, and (4) mature terminally differentiated endothelial cells (EC), which do not divide. Utilizing this assay, we compared the clonogenic potential of 1000 single adult human dermal microvascular endothelial cells (HMVECds), human umbilical vein endothelial cells (HUVECs), human umbilical artery endothelial cells (HUAECs), human coronary artery endothelial cells (HCAECs), and human aortic endothelial cells (HAECs) to the potential of adult peripheral and umbilical cord blood derived EPCs. We conducted four independent experiments. Remarkably, we demonstrate that a complete hierarchy of EPCs can be identified in EC populations derived from every vessel wall tested (Table I and n=4). Further, we show that ECs derived from each vessel wall cell population tested contain more proliferative EPCs (LPP-ECFCs and HPP-ECFCs) compared to EPCs derived from adult peripheral blood.Percent of 1,000 Single Cells PlatedMature ECEC-ClusterLPP-ECFCHPP-ECFCHUVEC42±618±229±911±5HAEC37±323±821±420±6HMVECd65±921±612±42±0.6HCAEC46±218±220±216±2HUAEC41±110±127±421±2Adult EPC81±99±112±80.2±0.2Cord EPC50±207±220±1023±9Thus, this study provides evidence that a diversity of EPCs exists in human vessels and provides a new conceptual framework for determining both the origin and function of EPCs in maintaining vessel integrity and contributing to new sites of angiogenesis.

Tissue Availability of Donor Mesenchymal Stem Cells Determines the Degree of Plasticity Following Transplantation in a Non-Injury Model.

The ability of bone marrow-derived Hematopoietic and Mesenchymal Stem Cells (MSC) to generate hepatocytes in a large non-injury sheep model of human stem cell transplantation has been demonstrated. Although in our sheep model the liver is still a hematopoietic organ at the time of transplantation, we hypothesized that the human BM-derived MSCs migrated preferentially to the BM, thus leaving few MSCs available to lodge in the liver and give rise to hepatocytes. Thus, in the present studies, we compared the ability and efficiency of clonally-derived BM MSC populations to give rise to liver cells upon either intra-hepatic (IH) or intra-peritoneal (IP) transplantation into fetal sheep. Five MSC clones were established from human adult BM by single cell deposition of Stro-1+ Gly-A-CD45- cells and expanded until sufficient cells were obtained. Phenotypic characterization demonstrated that all clones were positive for CD90, CD13, and CD29, and negative for hematopoietic markers such as CD34, CD133, and CD45. Five different clonal populations were used and each clone was transplanted either IP (n=7) or IH (n=6) into 55-60 days old fetal sheep recipients at 5x105cells/fetus. Evaluation of the recipients at 56-70 days post-transplant showed that clonally-derived MSC were not only able to generate hepatocytes, but they were also able to generate hematopoietic cells detectable both in the BM and in the PB. IP transplanted animals had a significantly higher level of human CD45+ in their BM (CD45=2.7±0.4%) compared to the IH transplanted animals (CD45=0.25±0.05%), demonstrating that IP injection favors the homing of these cells to the BM while upon IH injection, the human blood cells generated remain in circulation (2.6% CD45+). When we evaluated the presence of hematopoietic cells in the livers of these animals, the IP transplanted animals contained almost 2% CD45+ cells within their livers, while IH transplanted animals had levels of only 0.5-0.65%. When we looked at the generation of liver cells in both groups of animals by immunohistochemistry using monoclonal antibodies specific for human hepatocytes and albumin, we observed that IH injection was much more efficient at generating human hepatocytes than IP injection. The levels of human hepatocytes in most of the IH transplanted animals (10-15% positive cells) were roughly 5X higher than their IP counterparts (2.5-3%). Furthermore, human hepatocytes produced following IH injection were widely distributed throughout the liver parenchyma, while IP injection resulted in a periportal pattern of human hepatocytic activity. We also confirmed the functionality of the generated hepatocytes by detecting human albumin in circulation in these animals by ELISA. In conclusion, these studies show that in a non-injury model the number of available donor cells reaching the liver can be controlled by altering the route of administration, and that this availability determines both the levels of donor-derived hepatocytes generated and the pattern of distribution of these hepatocytes throughout the recipient liver.

The symmetry of initial divisions of human hematopoietic progenitors is altered only by the cellular microenvironment.

We examined if cellular elements or adhesive ligands were able to alter asymmetric divisions of CD34(+)/CD38(-) cells in contrast to soluble factors at a single cell level. After single cell deposition onto 96-well plates, cells were cocultured for 10 days with the stem cell supporting cell line AFT024, fibronectin (FN), or bovine serum albumin (BSA). The divisional history was monitored with time-lapse microscopy. Subsequent function for the most primitive cells was assessed using the myeloid-lymphoid-initiating cell (ML-IC) assay. Committed progenitors were measured using colony-forming cells (CFC). Only contact with AFT024 recruited significant numbers of CD34(+)/CD38(-) cells into cell cycle and increased asymmetric divisions. Although most ML-IC were still identified among cells that have divided fewer than 3 times, a significant number of ML-IC shifted into the fast-dividing fraction after exposure to AFT024. The increase in ML-IC frequency was predominantly due to recruitment of quiescent and slow-dividing cells from the starting population. Increase in CFC activity induced by AFT024 was found only among rapidly dividing cells. For the first time, we have demonstrated that asymmetric divisions can be altered upon exposure with a stem cell-supporting microenvironment. For the primitive subset of cells (ML-IC), this was predominantly due to recruitment into cell cycle and increased rounds of cycling without loss of function. Exposure to AFT024 cells also increased proliferation and asymmetric divisions of committed CFC. Hence direct communication between hematopoietic progenitors with stroma cells is required for maintaining self-renewal potential.

A method for clonal analysis of epidermal growth factor-responsive neural progenitors

Epidermal growth factor (EGF) responsive neural progenitors are defined by clonal growth from single cells. In previous studies we were unable to obtain clones at single cell densities using trypsinized cells and trituration alone always gave cellular aggregates. Here we report on single cell derived clones using a technique involving trituration of EGF responsive neurospheres, cell filtration, and single cell sorting using a MoFlo high speed fluorescence activated cell sorter. Single cell deposition was confirmed by labeling cells with Hoechst 33342 and Flow-check Fluorospheres, and visualization by fluorescence microscopy. The cells were deposited into liquid medium and grown from single cells in 10–20 ng/ml EGF for 12–14 days. This gave a cloning efficiency of 2.12%±0.37. New colonies occurred as late as day 18 post-sort. Tritiated thymidine suicide indicates that a percentage of these cells are cycling. Immunohistochemical analysis for oligodendrocytes, astroglia, and neuronal lineages performed on colonies at 10–14 and 21–28 days gave 39% uni-lineage, 36% bi-lineage, and 25% tri-lineage colonies. A total of five different types of progenitor cells were observed. In individual colonies, oligodendrons predominated with a lesser presence of astroglial or neuronal cell types. This approach establishes a reliable and reproducible method for single cell cloning of neurosphere cells.

Genomics and proteomics in cancer: Proceedings of the 3rd Samuel A. Latt conference, Detroit, Michigan

Genomics and proteomics in cancer: Proceedings of the 3^rd Samuel A. Latt conference, Detroit, Michigan

Neurotrophin channeling of neural progenitor cell differentiation

The act of defining neuropoietic progenitor/stem cells is still in its early phases. Epidermal growth factor (EGF) stimulates extended proliferation of aggregates of subventricular striatal cells, taken from E15 mouse striatum, termed neurospheres in liquid culture. We have shown here and in previous work, using either immunohistochemistry or RT-PCR, that neurosphere cells express 13 cytokines (32 tested) and 20 cytokine receptors (28 tested), with 11 potential paracrine and nine potential autocrine loops. The neurotrophin receptors, Trk A, B, and C, were all expressed. Using a newly developed FACS single cell deposition technique, we evaluated the capacity of single EGF stimulated neurosphere cells to respond to the ligands for Trk A and B, nerve growth factor (NGF), and brain-derived neurotrophin factor (BDNF). Addition of NGF or BDNF to EGF for 14 days had no effect, but removal of EGF at day 14 with subsequent addition of BDNF or NGF resulted in an increase in neuronal and astroglial, but not oligodendrocyte, colony cells at 21 and 28 days of culture for BDNF, and of both cell types at 28 days for NGF. Tri-lineage colonies increased at day 21 with BDNF and at day 28 for both NGF and BDNF. Gross colony morphology also showed changes with neurotrophin addition, forming multiple individual cell balls or filamentous spreads. When EGF was withdrawn, a threshold effect was observed, with small, but not large, colonies ceasing growth. BDNF and NGF showed no effects on cell proliferation when compared to EGF controls, as determined by 5'-bromo-2-deoxyuridine (BrdU) incorporation and thus, they appear to affect differentiation of progenitor cells. These data indicate a sequential action of cytokines with EGF maintaining viability and proliferation and blocking differentiation. Removal of EGF is then permissive for the differentiating effects of BDNF and NGF. These data further indicate that the majority of EGF neurosphere clones have neurotrophin dependent tri-lineage potential.

In Vitro Hematopoietic and Endothelial Cell Development From Cells Expressing TEK Receptor in Murine Aorta-Gonad-Mesonephros Region

Recent studies have shown that long-term repopulating hematopoietic stem cells (HSCs) first appear in the aorta-gonad-mesonephros (AGM) region. Our immunohistochemistry study showed that TEK+cells existed in the AGM region. Approximately 5% of AGM cells were TEK+, and most of these were CD34+ and c-Kit+. We then established a coculture system of AGM cells using a stromal cell line, OP9, which is deficient in macrophage colony-stimulating factor (M-CSF). With this system, we showed that AGM cells at 10.5 days postcoitum (dpc) differentiated and proliferated into both hematopoietic and endothelial cells. Proliferating hematopoietic cells contained a significant number of colony-forming cells in culture (CFU-C) and in spleen (CFU-S). Among primary AGM cells at 10.5 dpc, sorted TEK+ AGM cells generated hematopoietic cells and platelet endothelial cell adhesion molecule (PECAM)-1+ endothelial cells on the OP9 stromal layer, while TEK− cells did not. When a ligand for TEK, angiopoietin-1, was added to the single-cell culture of AGM, endothelial cell growth was detected in the wells where hematopoietic colonies grew. Although the incidence was still low (1/135), we showed that single TEK+ cells generated hematopoietic cells and endothelial cells simultaneously, using a single-cell deposition system. This in vitro coculture system shows that the TEK+ fraction of primary AGM cells is a candidate for hemangioblasts, which can differentiate into both hematopoietic cells and endothelial cells.

In Vitro Hematopoietic and Endothelial Cell Development From Cells Expressing TEK Receptor in Murine Aorta-Gonad-Mesonephros Region

AbstractRecent studies have shown that long-term repopulating hematopoietic stem cells (HSCs) first appear in the aorta-gonad-mesonephros (AGM) region. Our immunohistochemistry study showed that TEK+cells existed in the AGM region. Approximately 5% of AGM cells were TEK+, and most of these were CD34+ and c-Kit+. We then established a coculture system of AGM cells using a stromal cell line, OP9, which is deficient in macrophage colony-stimulating factor (M-CSF). With this system, we showed that AGM cells at 10.5 days postcoitum (dpc) differentiated and proliferated into both hematopoietic and endothelial cells. Proliferating hematopoietic cells contained a significant number of colony-forming cells in culture (CFU-C) and in spleen (CFU-S). Among primary AGM cells at 10.5 dpc, sorted TEK+ AGM cells generated hematopoietic cells and platelet endothelial cell adhesion molecule (PECAM)-1+ endothelial cells on the OP9 stromal layer, while TEK− cells did not. When a ligand for TEK, angiopoietin-1, was added to the single-cell culture of AGM, endothelial cell growth was detected in the wells where hematopoietic colonies grew. Although the incidence was still low (1/135), we showed that single TEK+ cells generated hematopoietic cells and endothelial cells simultaneously, using a single-cell deposition system. This in vitro coculture system shows that the TEK+ fraction of primary AGM cells is a candidate for hemangioblasts, which can differentiate into both hematopoietic cells and endothelial cells.

Single Adult Human CD34+/Lin−/CD38− Progenitors Give Rise to Natural Killer Cells, B-Lineage Cells, Dendritic Cells, and Myeloid Cells

AbstractMarrow stromal cultures support adult CD34+/Lin−/HLA-DR− or CD34+/Lin−/CD38− cell differentiation into natural killer (NK) or myeloid cells, but unlike committed lymphoid progenitors (CD34+/Lin−/CD45RA+/CD10+), no B cells are generated. We tested whether different microenvironments could establish a developmental link between the NK and B-cell lineages. Progenitors were cultured in limiting dilutions with interleukin-7 (IL-7), flt3 ligand (FL), c-kit ligand (KL), IL-3, IL-2, and AFT024, a murine fetal liver line, which supports culture of transplantable murine stem cells. NK cells, CD10+/CD19+ B-lineage cells and dendritic cells (DC) developed from the same starting population and IL-7, FL, and KL were required in this process. Single cell deposition of 3,872 CD34+/Lin−/CD38− cells onto AFT024 with IL-7, FL, KL, IL-2, and IL-3 showed that a one time addition of IL-3 at culture initiation was essential for multilineage differentiation from single cells. Single and double lineage progeny were frequently detected, but more importantly, 2% of single cells could give rise to at least three lineages (NK cells, B-lineage cells, and DC or myeloid cells) providing direct evidence that NK and B-lineage differentiation derive from a common lymphomyeloid hematopoietic progenitor under the same conditions. This study provides new insights into the role of the microenvironment niche, which governs the earliest events in lymphoid development.

Single Adult Human CD34+/Lin−/CD38− Progenitors Give Rise to Natural Killer Cells, B-Lineage Cells, Dendritic Cells, and Myeloid Cells

Marrow stromal cultures support adult CD34+/Lin−/HLA-DR− or CD34+/Lin−/CD38− cell differentiation into natural killer (NK) or myeloid cells, but unlike committed lymphoid progenitors (CD34+/Lin−/CD45RA+/CD10+), no B cells are generated. We tested whether different microenvironments could establish a developmental link between the NK and B-cell lineages. Progenitors were cultured in limiting dilutions with interleukin-7 (IL-7), flt3 ligand (FL), c-kit ligand (KL), IL-3, IL-2, and AFT024, a murine fetal liver line, which supports culture of transplantable murine stem cells. NK cells, CD10+/CD19+ B-lineage cells and dendritic cells (DC) developed from the same starting population and IL-7, FL, and KL were required in this process. Single cell deposition of 3,872 CD34+/Lin−/CD38− cells onto AFT024 with IL-7, FL, KL, IL-2, and IL-3 showed that a one time addition of IL-3 at culture initiation was essential for multilineage differentiation from single cells. Single and double lineage progeny were frequently detected, but more importantly, 2% of single cells could give rise to at least three lineages (NK cells, B-lineage cells, and DC or myeloid cells) providing direct evidence that NK and B-lineage differentiation derive from a common lymphomyeloid hematopoietic progenitor under the same conditions. This study provides new insights into the role of the microenvironment niche, which governs the earliest events in lymphoid development.

Progressive lineage analysis by cell sorting and culture identifies FLK1+VE-cadherin+ cells at a diverging point of endothelial and hemopoietic lineages.

Totipotent murine ES cells have an enormous potential for the study of cell specification. Here we demonstrate that ES cells can differentiate to hemopoietic cells through the proximal lateral mesoderm, merely upon culturing in type IV collagen-coated dishes. Separation of the Flk1+ mesoderm from other cell lineages was critical for hemopoietic cell differentiation, whereas formation of the embryoid body was not. Since the two-dimensionally spreading cells can be monitored easily in real time, this culture system will greatly facilitate the study of the mechanisms involved in the cell specification to mesoderm, endothelial, and hemopoietic cells. In the culture of ES cells, however, lineages and stages of differentiating cells can only be defined by their own characteristics. We showed that a combination of monoclonal antibodies against E-cadherin, Flk1/KDR, PDGF receptor(alpha), VE-cadherin, CD45 and Ter119 was sufficient to define most intermediate stages during differentiation of ES cells to blood cells. Using this culture system and surface markers, we determined the following order for blood cell differentiation: ES cell (E-cadherin+Flk1-PDGFRalpha-), proximal lateral mesoderm (E-cadherin-Flk1+VE-cadherin-), progenitor with hemoangiogenic potential (Flk1+VE-cadherin+CD45-), hemopoietic progenitor (CD45+c-Kit+) and mature blood cells (c-Kit-CD45+ or Ter119+), though direct differentiation of blood cells from the Flk1+VE-cadherin- stage cannot be ruled out. Not only the VE-cadherin+CD45- population generated from ES cells but also those directly sorted from the yolk sac of 9.5 dpc embryos have a potential to give rise to hemopoietic cells. Progenitors with hemoangiogenic potential were identified in both the Flk1+VE-cadherin- and Flk1+VE-cadherin+ populations by the single cell deposition experiment. This line of evidence implicates Flk1+VE-cadherin+ cells as a diverging point of hemopoietic and endothelial cell lineages.

Improved differential screening approach to analyse transcriptional variations in organized cDNA libraries

A cDNA library was generated from rat brain tissues and organized into 1536-well plates, using a fluorescence activated cell sorter (FACS), acting as a single cell deposition system. The organized library containing 10 000 clones, with 60% full-length cDNA inserts, allowed the generation of multiple identical membrane replicas. Each replica was hybridized with a complex probe obtained from a particular brain tissue or a given cultured cell. The signal intensity for each of the clones present on the membrane, quantified with a standard image-analysis software, is proportional both to the abundance of the corresponding mRNA in the probe and to the amount of plasmid template on the membrane. The latter value was thus used to normalize the signals produced with complex probes, to optimize the comparison of mRNA expression levels for the different systems under study. The construction of high-quality cDNA libraries, the generation of identical membrane replicas and comparable probes, and the utilization of an image-analysis software package, coupled with the normalization of the spot intensity by assaying plasmid quantity, significantly improves the differential screening approach. Altogether, these technical improvements open the possibility to compare a great number of different probes and, in consequence, to accumulate biological information for each clone present in an organized cDNA library. The functional information obtained should complement data from DNA sequencing projects.

Selective cloning of hybridoma cells for enhanced immunoglobulin production using flow cytometric cell sorting and automated laser nephelometry

Techniques for selective cloning of murine hybridoma cells by flow cytometric cell sorting and use of automated laser nephelometry to determine the resultant clones' immunoglobulin secretion levels are described. Using a commercially available attachment to a fluorescence-activated cell sorter, individual hybridoma cells were successfully distributed into microtiter wells in an automated manner based on their forward angle light scatter properties and their reaction to fluorescein-conjugated anti-mouse-IgG. The techniques were used to estimate successfully the frequency of immunoglobulin-secreting cells in established cultures. In addition, heterogeneity of cell surface immunoglobulin expression was observed and utilized as a criterion for flow sorting of new hybridoma variants. In these studies, clones derived from high (anti-IgG) intensity sorting regions yielded cultures with enhanced immunoglobulin secretion levels, as determined by automated laser nephelometry. Furthermore, the surface immunoglobulin phenotype of the derived clones was conserved in subsequent progeny. Finally, it was established that inclusion of propidium iodide in the hybridoma cell sorting mixtures improved cloning efficiency by facilitating enhanced discrimination and elimination of nonviable cells. Our results indicate that flow cytometric-assisted single cell deposition provides positive attributes of several traditional hybridoma cloning techniques and, in addition, furnishes a tool for steering the cloning process toward selection of enhanced immunoglobulin producing cultures.

Clonal analysis of HLA-DR and -DQ associated determinants: Their contribution to Dw specificities

In order to investigate the distribution of epitopes recognized by T-cell clones directed against HLA class II products, bulk primed cell populations were generated using cells matched for class I determinants but disparate for class II determinants. Cells were cloned by single cell deposition (FACS IV) or limiting dilution (1 cell/3 wells), and assayed for proliferative and cytolytic function with panels of well-characterized cells. All cytolytic clones generated from an anti-DR4/Dw4/DQw3 priming combination or an anti-DR2/Dw2/DQw1 priming combination lysed essentially all targets sharing the same Dw type as the sensitizing cell. In some cases, other targets were also lysed. For instance, some clones were lytic to targets bearing the same DR antigen but another Dw subtype including a few clones lytic to virtually all cells carrying that DR specificity. An occasional target cell expressing a different DR antigen from the sensitizing cell was also lysed by these clones, in some cases to the same extent of lysis seen on the specific target. Monoclonal antibody inhibition studies identified three groups of clones: the DQ directed clones and clones apparently directed at more than one DR product. However, the number of molecules detected for each haplotype remains to be investigated. Our data indicate that determinants detected on both DR and DQ products are associated with the Dw type of the sensitizing cell showing that there is polymorphism recognized by T cells on both DR and DQ that is subtypic to the serologically defined specificities. Thus, it appears that the bulk T-cell response is a composite of individual clones recognizing distinct determinants on these class II molecules. The implications of these findings for studies of HLA restricted recognition are discussed.

Analysis of human class II antigens by cloned cytolytic T cell reagents: A study using HLA loss mutant lymphoblastoid cell lines and monoclonal antibodies detecting the HLA-DP product(s)

We have utilized cloned T cell reagents and ionizing radiation-induced mutants of an HLA heterozygous lymphoblastoid cell line (LCL) to investigate the determinants detected by the cell-mediated lympholysis (CML) assay. Cells of an LCL clone, 721.501, and HLA haplotype loss mutant expressing the HLA-A2-Cw1-Bw51-DR1-Dw1-DQw1-DPw2-GLO haplotype were used as sensitizing cells for responder cells in vitro. “Cloned” reagents were generated by single-cell deposition of cells of a bulk reagent primed against 721.501 cells. Those clones were screened for cytolytic activity against HLA loss mutant targets (derived from LCL 721) of four different categories; HLA-A2 loss only. A2-Cw1-Bw51 loss, A2-Cw1-Bw51-DR1-DQw1 loss, and the entire HLA haplotype loss. Of 196 clones tested, 36 were cytolytic, including three anti-A2, five anti-Bw51/Cw1, 12 anti-DR1/DQw1, 13 anti-DP region associated with DPw2, and three of undetermined specificity, based on cytolytic patterns against the HLA loss mutant targets. Of 25 anti-HLA class II lytic clones, 23 (92%) fitted the cahracteristics of helper cell-independent cytolytic T cells (HITc), whereas only two of eight (25%) anti-class I clones were HITc. The 13 anti-DP region clones were divided into three subgroups defined by blocking by anti-FA and not Tü39 monoclonal antibodies (MoAb), by Tü39 and not anti-FA, and by both MoAbs.