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PubMed2006

Constitutively active beta-catenin promotes expansion of multipotent hematopoietic progenitors in culture.

This study was designed to investigate one component of the Wnt/beta-catenin signaling pathway that has been implicated in stem cell self-renewal. Retroviral-mediated introduction of stable beta-catenin to primitive murine bone marrow cells allowed the expansion of multipotential c-Kit(low)Sca-1(low/-)CD19(-) CD11b/Mac-1(-)Flk-2(-)CD43(+)AA4.1(+)NK1.1(-)CD3(-)CD11c(-)Gr-1(-)CD45R/B220(+) cells in the presence of stromal cells and cytokines. They generated myeloid, T, and B lineage lymphoid cells in culture, but had no T lymphopoietic potential when transplanted. Stem cell factor and IL-6 were found to be minimal requirements for long-term, stromal-free propagation, and a beta-catenin-transduced cell line was maintained for 5 mo with these defined conditions. Although multipotential and responsive to many normal stimuli in culture, it was unable to engraft several types of irradiated recipients. These findings support previous studies that have implicated the canonical Wnt pathway signaling in regulation of multipotent progenitors. In addition, we demonstrate how it may be experimentally manipulated to generate valuable cell lines.

Animals↗
PubMed1995

Patterns of expression of lineage-specific markers during the in vitro-induced differentiation of HT29 colon carcinoma cells.

The four different cell types present in the mature colon, absorptive enterocytes, mucus-secreting goblet cells, Paneth cells, and enteroendocrine cells, are believed to derive from a common precursor, the stem cell, anchored near the base of the crypt. Stem cell descendants undergo several rounds of cell division, creating a pool of transit cells that are committed to differentiation. The mechanisms by which committed cells are allocated to the different cell lineages of the intestine are poorly understood. We have used the colon carcinoma cell line HT29 and Cl.16E cells, a clonal derivative of HT29 cells, to investigate the regulation and pattern of expression of several markers (MUC2, MUC3, carcinoembryonic antigen, and alkaline phosphatase) that are associated with a more differentiated phenotype and that, in the mature cells, are lineage restricted. HT29 cells can express, upon exposure to the appropriate inducers, distinct intestinal specific markers; they are, therefore, considered multipotent, similar to the stem cells of the crypt. Conversely, Cl.16E cells are lineage restricted and respond to cell contact inhibition by expressing a fully differentiated goblet cell phenotype. We show that, in HT29 cells, different inducers (12-O-tetradecanoylphorbol-13-acetate, forskolin, and sodium butyrate) modulate specific sets of markers. Forskolin induces the expression of both MUC2 and MUC3, whereas 12-O-tetradecanoylphorbol-13-acetate is capable of inducing only MUC2, and sodium butyrate, only MUC3 gene expression. Carcinoembryonic antigen, a marker common to enterocytes and goblet cells; can be induced by all the agents, whereas the alkaline phosphatase gene, the expression of which is characteristic of enterocytes, is responsive solely to sodium butyrate treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed2002

MLL-ENL cooperates with SCF to transform primary avian multipotent cells.

The MLL gene is targeted by chromosomal translocations, which give rise to heterologous MLL fusion proteins and are associated with distinct types of acute lymphoid and myeloid leukaemia. To determine how MLL fusion proteins alter the proliferation and/or differentiation of primary haematopoietic progenitors, we introduced the MLL-AF9 and MLL-ENL fusion proteins into primary chicken bone marrow cells. Both fusion proteins caused the sustained outgrowth of immature haematopoietic cells, which was strictly dependent on stem cell factor (SCF). The renewing cells have a long in vitro lifespan exceeding the Hayflick limit of avian cells. Analysis of clonal cultures identified the renewing cells as immature, multipotent progenitors, expressing erythroid, myeloid, lymphoid and stem cell surface markers. Employing a two-step commitment/differentiation protocol involving the controlled withdrawal of SCF, the MLL-ENL-transformed progenitors could be induced to terminal erythroid or myeloid differentiation. Finally, in cooperation with the weakly leukaemogenic receptor tyrosine kinase v-Sea, the MLL-ENL fusion protein gave rise to multilineage leukaemia in chicks, suggesting that other activated, receptor tyrosine kinases can substitute for ligand-activated c-Kit in vivo.

Animals↗
PubMed2002

Expansion of mammalian neural stem cells in bioreactors: effect of power input and medium viscosity.

Multipotent neural precursors can be cultured in suspension bioreactors as aggregates of stem cells and progenitor cells. However, it is important to limit the size of the aggregates, as necrotic centers may develop at very large diameters. Previously, we have shown that the hydrodynamics within a suspension bioreactor can be used to control the diameter of NSC aggregates (D(MAVG)<150 microm) below sizes where necrosis would be expected to occur. In the present study, power law correlations were developed for our bioreactors showing the dependence of the maximum mean aggregate diameter on both the kinematic viscosity of the medium and the power input per unit mass of medium. The power input was manipulated by changing the agitation rate (60-100 rpm), and the viscosity was manipulated through the addition of non-toxic levels of carboxymethylcellulose. The study also confirmed that the maximum liquid shear generated at the surface of the aggregates was sufficient to dislodge single cells, thus limiting the maximum diameter of the aggregates, without causing cell damage (tau(max)=9.76 dyn/cm(2)). This is a first step in the development of a reproducible, scaled-up process for the production of neural stem cells for therapeutic applications including the treatment of neurodegenerative disorders and acute central nervous system injuries.

Animals↗
PubMed2006

Characterization of four clones derived from human adenocarcinoma cell line, HT29, and analysis of their response to sodium butyrate.

The differentiation of colorectal cancer cells is associated with the arrest of tumor growth and tumor regression. However, the mechanism of such tumor cell differentiation has not yet been elucidated. Several adenocarcinoma cell lines, including HT29 which differentiates only upon stimulation with a differentiation agent, have been used for the study of colorectal cells. Since we had previously obtained variable results during analyses of these cells, we selected several clones of this cell line. In this study, four clones of the parental HT29 cells, H8, G9, G10 and A3, were characterized. All of them differentiated upon treatment with sodium butyrate as the differentiation agent but they appeared different in their response regarding some of the markers of differentiation. As revealed by ultrastructural analysis, H8 and G10 clones formed numerous intercellular cysts with microvilli whereas these structures were found only occasionally in G9 and A3 clones. An elevated level of the indicator of cell differentiation, CEACAM 1, was found in H8 and G10 clones and the activity of alkaline phosphatase, another important marker of colorectal cell differentiation, was up-regulated and highly increased upon butyrate treatment in the H8 clone. Phosphorylation of p38 MAPK was increased in H8 and A3 butyrate-treated clones. According to the levels of cleaved PARP and activated caspase-3, the apoptotic response to butyrate appeared similar in all four clones, while electronoptic analysis revealed that clones G9 and A3 were more perceptive to butyrate-induced apoptosis. In conclusion, our data showed considerable heterogeneity in morphology and some enzymatic activity of the cloned cells. This fact may contribute to the evidence that many HT29 cells possess multipotent information similar to that of stem cells of the normal intestinal crypt.

Adenocarcinoma↗
PubMed2005

Hematopoietic differentiation of human embryonic stem cells progresses through sequential hematoendothelial, primitive, and definitive stages resembling human yolk sac development.

We elucidate the cellular and molecular kinetics of the stepwise differentiation of human embryonic stem cells (hESCs) to primitive and definitive erythromyelopoiesis from human embryoid bodies (hEBs) in serum-free clonogenic assays. Hematopoiesis initiates from CD45 hEB cells with emergence of semiadherent mesodermal-hematoendothelial (MHE) colonies that can generate endothelium and form organized, yolk sac-like structures that secondarily generate multipotent primitive hematopoietic stem progenitor cells (HSPCs), erythroblasts, and CD13+CD45+ macrophages. A first wave of hematopoiesis follows MHE colony emergence and is predominated by primitive erythropoiesis characterized by a brilliant red hemoglobinization, CD71/CD325a (glycophorin A) expression, and exclusively embryonic/fetal hemoglobin expression. A second wave of definitive-type erythroid burst-forming units (BFU-e's), erythroid colony-forming units (CFU-e's), granulocyte-macrophage colony-forming cells (GM-CFCs), and multilineage CFCs follows next from hEB progenitors. These stages of hematopoiesis proceed spontaneously from hEB-derived cells without requirement for supplemental growth factors during hEB differentiation. Gene expression analysis of differentiating hEBs revealed that initiation of hematopoiesis correlated with increased levels of SCL/TAL1, GATA1, GATA2, CD34, CD31, and the homeobox gene-regulating factor CDX4 These data indicate that hematopoietic differentiation of hESCs models the earliest events of embryonic and definitive hematopoiesis in a manner resembling human yolk sac development, thus providing a valuable tool for dissecting the earliest events in human HSPC genesis.

Antigens, CD↗
PubMed2001

Theory of robustness of irreversible differentiation in a stem cell system: chaos hypothesis.

Based on an extensive study of a dynamical systems model of the development of a cell society, a novel theory for stem cell differentiation and its regulation is proposed as the "chaos hypothesis". Two fundamental features of stem cell systems-stochastic differentiation of stem cells and the robustness of a system due to regulation of this differentiation-are found to be general properties of a system of interacting cells exhibiting chaotic intra-cellular reaction dynamics and cell division, whose presence does not depend on the detail of the model. It is found that stem cells differentiate into other cell types stochastically due to a dynamical instability caused by cell-cell interactions, in a manner described by the Isologous Diversification theory. This developmental process is shown to be stable not only with respect to molecular fluctuations but also with respect to the removal of cells. With this developmental process, the irreversible loss of multipotency accompanying the change from a stem cell to a differentiated cell is shown to be characterized by a decrease in the chemical diversity in the cell and of the complexity of the cellular dynamics. The relationship between the division speed and the loss of multipotency is also discussed. Using our model, some predictions that can be tested experimentally are made for a stem cell system.

Animals↗
PubMed2000

In vitro differentiation of multipotent human neural progenitors in serum-free medium.

Stem cells are exciting candidates for therapeutic strategies in neurodegenerative diseases, due to their multipotency and migratory capabilities. We show that stem cell-like embryonic normal human neural progenitors (HNPs) are capable of proliferating in response to mitogenic growth factors and differentiate into diverse CNS cell types in vitro. We present evidence that HNPs differentiate to beta III-tubulin-, glial fibrillary acidic protein- and O4-immuno-positive cells, in both a fetal bovine serum-containing and a non-supplemented, serum-free basal medium. From these findings, we propose that HNPs may differentiate not only in response to exogenous differentiation factor(s) contained in the serum, but also in response to some endogenous factor(s) released from the HNPs, which may regulate the differentiation pathway of these cells.

Antigens, Differentiation↗
PubMed1994

The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype.

The Thy-1.1loSca-1hiLin-/lo population, representing 0.05% of C57BL/Ka-Thy-1.1 bone marrow, is highly enriched for hematopoietic stem cells and includes all multipotent progenitors in this mouse strain; however, the functional reconstituting activity of this fraction is heterogeneous. Only around 25% of clonal reconstitutions by cells from this population are long term; remaining clones yield transient multilineage reconstitutions. By fractionating based on lineage marker expression, the Thy-1.1loSca-1hiLin-/lo population has been resolved into three subpopulations: Lin-Mac-1-CD4-; Lin-Mac-1loCD4-; and Mac-1loCD4lo. Of these, only the Lin-Mac-1-CD4- population is highly enriched for long-term reconstituting hematopoietic stem cells. A comparison of transient and long-term multipotent progenitors indicates that long-term progenitors have less CFU-S activity, are equally radioprotective, and are less frequently in cell cycle. The ability to predict the longevity of reconstitution based on lineage marker expression indicates that reconstitution potential is deterministic, not stochastic.

Animals↗
PubMed1992

Expression and downregulation of cytotoxic cell protease 1 or Granzyme 'B' transcripts during myeloid differentiation of interleukin-3-dependent murine stem cell lines.

Using the technique of differential cDNA library screening, we have molecularly cloned a gene that is highly expressed in an undifferentiated myeloid multipotent and growth factor-dependent stem cell line (FDCP-Mix) and that downregulates as these cells are induced to differentiate along monocytic, granulocytic, and erythroid cell lineages. Sequence analysis of this gene has shown homology with a previously cloned gene, cytotoxic cell protease 1 (CCP1 or Granzyme 'B'), that has been shown to be expressed only in thymocytes, activated T cells, a mast cell line, and peritoneal exudate leukocytes. In situ hybridization, Northern blot analysis, and nuclear run-off assay has confirmed that expression of CCP1 is restricted to the phenotypically primitive multipotent undifferentiated. FDCP-Mix cells that are undergoing self-renewal in the presence of growth factors such as interleukin-3.

Amino Acid Sequence↗
PubMed1996

Central nervous system cell transplantation: a novel therapy for storage diseases?

Many methods of gene transfer to the brain are under study. One employs a neural stem cell based strategy. Transplanting neural progenitor cells that intrinsically secrete missing or therapeutic gene products, or are genetically engineered ex vivo to do so, may provide a strategy for long-term treatment of central nervous system manifestations of a number of neurogenetic diseases. Multipotent neural progenitors or stem cells (or cells that mimic their behavior) are capable of differentiating along multiple central nervous system cell-type lineages. They can engraft as integral members of normal structures throughout the host central nervous system without disturbing other neurobiological processes. They can also be easily genetically manipulated ex vivo. By exploiting their basic biological properties, these cells may be able to deliver therapeutic gene products in a sustained, direct, and perhaps regulated fashion throughout the central nervous system. Furthermore, although they may disseminate these gene products throughout the brain, they nevertheless restrict that distribution to only the central nervous system. Thus, these vehicles may overcome many of the limitations of viral and non-neural cellular vectors, as well as pharmacologic and genetic interventions. The feasibility of this neural stem cell-based strategy has been demonstrated by correcting the widespread central nervous system pathology of a murine model of a prototypical inherited neurodegenerative disease, mucopolysaccharidosis type VII. These studies have helped to establish the use of such cells as a paradigm for transferring other molecules of therapeutic or developmental interest throughout the central nervous system at many ages.

Animals↗
PubMed2001

Evidence for stem-cell niches in the tracheal epithelium.

It is generally important to elucidate airway epithelial cell lineages and to identify multipotent progenitors as targets for gene therapy. Stem (S) cells are typically present in specialized compartments spatially proximal to their differentiated progeny, but an equivalent paradigm has not been demonstrated in the airway. We discovered a distinct population of cells displaying high levels of keratin expression in murine tracheal submucosal gland ducts, and tested the hypothesis that bromodeoxyuridine (BrdU) label-retaining cells (LRCs), thought to represent the S-cells, were present in this compartment. Mice received weekly epithelial damage by intratracheal detergent or SO(2) inhalation for 4 wk and received intraperitoneal injections of BrdU every 48 h during the injury and repair period. At 3 and 6 d after injury, BrdU-positive epithelial cells were noted along the entire tracheal length in both basal and lumenal cell positions. At later time points (20 and 95 d) LRCs were localized to gland ducts in the upper trachea and to systematically arrayed foci in the lower trachea, typically near the cartilage-intercartilage junction. LRCs were not pulmonary neuroendocrine cells. Heterotopic tracheal grafts after surface epithelial removal demonstrated reconstitution of a surface-like epithelium from gland remnants. These results suggest that airway epithelial S cells are localized to specific niches.

Animals↗
PubMed2003

MLL-GAS7 transforms multipotent hematopoietic progenitors and induces mixed lineage leukemias in mice.

A specific association with mixed lineage leukemias suggests that MLL oncoproteins may selectively target early multipotent hematopoietic progenitors or stem cells. We demonstrate here that a representative MLL fusion protein, MLL-GAS7, impairs the differentiation and enhances the in vitro growth of murine hematopoietic cells with multipotent features. The multilineage differentiation potential of these cells was suggested by their immuno-phenotypes and transcriptional programs and confirmed by their ability to induce three pathologically distinct leukemias in mice, including an acute biphenotypic leukemia (ABL) that recapitulates the distinctive hallmark features of many MLL-associated leukemias in humans. This experimental modeling of ABL in mice highlights its origin from multipotential progenitors that arrest at a bipotential stage specifically targeted or induced by MLL oncogenes.

Animals↗
PubMed2005

Definitive hematopoiesis from acetyl LDL incorporating endothelial cells in the mouse embryo.

Previously, we reported generation of erythropoiesis from acetyl low-density lipoprotein (Ac-LDL)- incorporating endothelial cells (ECs) in the mouse embryo. However, it is still unclear whether the other types of definitive hematopoietic cells (HCs) can be generated from these cells. In this study, ECs at 10 days post coitum (dpc) were tagged with Ac-LDL-DiI and were shown to release DiI+ HCs into the circulation after 12 h of whole embryo culture. The hematopoietic clusters in the main arteries were also stained with DiI. The circulating DiI+ HCs expressed c-Kit and half of these cells displayed blastic morphology. In vitro culture and in vivo reconstitution experiments demonstrated that the circulating DiI+ HCs contained definitive multipotent progenitors, including hematopoietic stem cells (HSCs). Furthermore, the sorted DiI+ HCs were able to colonize the fetal liver (FL) when introduced back into the blood stream of a secondary recipient embryo. These results suggest that Ac-LDL incorporating ECs can produce definitive HSCs and HCs colonizing FL in the mouse embryo.

Animals↗
PubMed2000

Excitable properties in astrocytes derived from human embryonic CNS stem cells.

Although it is widely believed that astrocytes lack excitability in adult tissue, primitive action potential-like responses have been elicited from holding potentials negative to -80 mV, in cultured and injury-induced gliotic rodent astrocytes and in human glia under pathological conditions such as glioblastomas and temporal lobe epilepsy. The present study was designed to investigate the properties of astrocytes (identified by immunoreactivity for glial fibrillary acidic protein) derived from multipotent human embryonic CNS stem cells and cultured for 12-25 days in differentiating conditions. We describe here for the first time that brief (1 ms) current pulses elicit spikes from a resting potential (VREST) of approximately -37 mV and, more interestingly, that spontaneous firing can be occasionally recorded in human astrocytes. A voltage-clamp study revealed that in these cells: (i) the half-inactivation of the tetrodotoxin (TTX)-sensitive Na+ channels is around VREST; (ii) the delayed rectifier K+ current is very small; (iii) the ever-present transient outward A-type K+ channels are paradoxically capable of inhibiting the action potentials elicited from a negative membrane potential (-55 to -60 mV); and (iv) inwardly rectifying currents are not present. The responses predicted from a simulation model are in agreement with the experiments. As suggested by recent studies, the decrease of Na+ channel expression and the changes of the electrophysiological properties during the postnatal maturation of the CNS seem to exclude the possibility that astrocytes may play an excitable role in adult tissue. Our data show that excitability and firing should be considered an intrinsic attribute of human astrocytes during CNS development. This is likely to have physiological importance because the role of astrocytes during development is different from the [K+]o-buffering role played in adult CNS, namely the glutamate release and/or the guiding of migrating neurons.

4-Aminopyridine↗
PubMed1979

Granulocytic chalone inhibits rapidly proliferating committed murine progenitor cells (CFU-C).

Bovine leucocyte extract containing the granulocytic chalone inhibited proliferation of rapidly, but not slowly, prliferating murine CFU-C. The precursors of CFU-C, i.e. multipotent colon-forming stem cells (CFU-S), were apparently not affected. Inhibition of proliferation was detected as a decreased killing of DNA synthesizing cells in diffusion chamber cultures by hydroxyurea, after pre-treatment with the extract.

Animals↗
PubMed2003

Hematopoietic stem cell emergence in the human embryo and fetus.

Two waves of hematopoietic stem cell generation take place in the first month of human gestation. The first one has long been known to occur in the yolk sac; only recently was another one identified that results in the development, from the 27th day, of clusters of hematopoietic cells on the ventral endothelium of the aorta and vitelline artery. This latter, intra-embryonic phase of blood cell progenitor production is undoubtedly local since its presumptive territory of occurrence, the para-aortic splanchnopleura, is endowed with blood-forming potential in vitro from, at least, day 19 of development. The first multipotent, lympho-myeloid stem cells emerge within that intra-embryonic territory, whereas the yolk sac only produces myeloid precursor cells devoid of lymphoid potential. The forerunners of the hematopoietic stem cells born to intra-embryonic arteries appear to migrate from the splanchnopleura as KDR(+) angio-hematopoietic cells that colonize the ventral wall of the aorta to give rise to hematogenous endothelium. All these results indicate that stem cells for human definitive hematopoiesis emerge within the embryo, in the walls of truncal arteries, from splanchnopleural ancestors and through a vascular endothelial intermediate stage. Less expectedly, blood-forming endothelial cells were also encoutered in the embryonic and fetal liver and in the fetal and, even, adult bone marrow, suggesting that a filiation between vascular and hematopoietic cells may persist during the whole life.

Antigens, CD34↗
PubMed2006

Human progenitor cells isolated from the developing cortex undergo decreased neurogenesis and eventual senescence following expansion in vitro.

Isolation of a true self-renewing stem cell from the human brain would be of great interest as a reliable source of neural tissue. Here, we report that human fetal cortical cells grown in epidermal growth factor expressed low levels of telomerase and telomeres in these cultures shortened over time leading to growth arrest after 30 weeks. Following leukemia inhibitory factor (LIF) supplementation, growth rates and telomerase expression increased. This was best demonstrated following cell cycle synchronization and staining for telomerase using immunocytochemistry. This increase in activity resulted in the maintenance of telomeres at approximately 7 kb for more than 60 weeks in vitro. However, all cultures displayed a lack of oligodendrotye production, decreases in neurogenesis over time and underwent replicative senescence associated with increased expression of p21 before 70 weeks in vitro. Thus, under our culture conditions, these cells are not stable, multipotent, telomerase expressing self-renewing stem cells. They may be more accurately described as human neural progenitor cells (hNPC) with limited lifespan and bi-potent potential (neurons/astrocytes). Interestingly, hNPC follow a course of proliferation, neuronal production and growth arrest similar to that seen during expansion and development of the human cortex, thus providing a possible model neural system. Furthermore, due to their high expansion potential and lack of tumorogenicity, these cells remain a unique and safe source of tissue for clinical transplantation.

Cell Culture Techniques↗