Stochastic model for multipotent hemopoietic progenitor differentiation

In this study, the authors propose a stochastic model for multipotent hemopoietic progenitor differentiation, which assumes that there is a fixed probability (P) that a progenitor with a potential for differentiation along a particular lineage maintains the potential in each cell division in each daughter cell, and this differentiation process of each lineage proceeds independently. To examine the applicability of this model, a sequential micromanipulation of paired progenitors was carried out and followed by cytological examination of the cells contained in the colonies derived from these progenitors; then calculation was made of the ratio of the number of paired colonies containing cell(s) with a particular lineage to the number of paired colonies in which only one colony contained cell(s) with the lineage at the first and second cell division. The ratios were similar at the first and second cell division within each lineage. Furthermore, the frequences of each lineage in multilineage hemopoietic colonies were calculated using the P values obtained from these micromanipulation experiments. The expected frequencies were similar to those in the actual experiments. These results suggested that the stochastic model was applicable to multipotent hemopoietic progenitor differentiation.     

Stochastic compartmental model with branching particles

A multi-compartmental model with particles producing offspring according to the Markov branching process has been studied. Explicit results are given for the two-compartmental system and for irreversible general multicompartmental systems. The known models in stochastic compartmental analysis are shown to be particular cases of this model and applications are cited.     

Regulation of hemopoietic cell differentiation and proliferation

Differentiation and proliferation of almost all hemopoietic cell lines can now be studied in vitro. Cloning techniques and suspension cultures allow the study of proliferation of the multipotential hemopoietic progenitor cell and the committed progenitors for granulocytes, macrophages, eosinophils, megakryocytes, and erythrocytes. The proliferation of each of the committed progenitor cells is controlled by specific glycoproteins and two of these have recently been purified: granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin. The rate of proliferation of the GM-progenitor cells and their pattern of differentiation depends on the concentration of the hormone. At low concentrations of GM-CSF (10^?11 M) fewer progenitor cells are stimulated and macrophage colonies rather than granulocyte colonies develop. The change in the direction of granulocyte-macrophage differentiation appears to be related to (a) the concentration of GM- CSF and (b) the different sensitivity of a subpopulation of monocyte colony-forming cells which are responsive to GM-CSF even at low concentrations of the regulator. Analysis of the rate of RNA synthesis by bone marrow cells has shown that GM-CSF stimulates the mature nondividing end cells of differentiation (ie, polymorphs) as well as the progenitor cells. Although GM-CSF and erythropoietin have been radiolabeled, binding studies have been hampered by the loss of biologic activity during the labeling procedure and the heterogeneity of the target cells to which the regulators bind. Surface proteins and receptors for erythrocytes have been well characterized but the relationships between these proteins and the cell surface proteins of nucleated blood cells is not well understood. It appears that some proteins are lost from the cell surface during the development of granulocytes, which are retained on the surface of the B lymphocyte. Other proteins such as chemotactic receptors and complement receptors only appear on the mature cells. External radiolabeling of the granulocyte surface using iodogen yielded a simple profile of ¹²⁵I-labeled proteins when analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis.     

Recent Australian experience with hemopoietic stem and progenitor cell expansion

This review provides insight into two clinical trials conducted with ex vivo manipulated CD34+ cells. The first was an attempt to deliver a gene therapy for treatment of HIV and the second an attempt to improve rates of hemopoietic recovery with ex vivo generated myeloid cells.     

Multi-type branching models to describe cell differentiation programs

Cell proliferation and differentiation is described by a multi-type branching process, a probability model that defines the inheritance of cell type. Cell type is defined by (i) a repression index related to the time required for S-phase entry and (ii) phenotype as determined by cell markers and division history. The inheritance of cell type is expressed as the expected number and type of progeny cells produced by a mother cell given her type. Expressions for the expected number and type of cells produced by a multi-cellular (bulk culture) system are derived from the general model by making the simplifying assumption that cell generation times are independent. The multi-type Smith-Martin model (MSM) makes the further assumption that cell generation times are lag-exponentially distributed with phenotype transitions occurring just before entry into S-phase. The inheritance-modified MSM (IMSM) model includes the influence of generation time memory so that mother and daughter generation times are correlated. The expansion of human cord blood CD34⁺ cells by haematopoietic growth factors was division tracked in bulk culture using carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE). The MSM model was fitted to division tracking data to indentify cell cycle length, and the rates of CD34 antigen down-regulation and apoptosis. The IMSM model was estimated for mouse granulocyte-macrophage progenitors using live cell imaging data. Multi-type branching models describe cell differentiation dynamics at both single- and multi-cell scales, providing a new paradigm for systematic analysis of stem and progenitor cell development.     

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.     

Interaction of serum and colony-stimulating factor for survival of a factor-dependent hemopoietic progenitor cell line

The growth in vitro of the murine myeloid cell line FDC-P1 depends on the presence of serum and a murine hemopoietic growth factor (either granulocyte/macrophage colony-stimulating factor (GM-CSF) or multipotential colony-stimulating factor (multi-CSF, IL3]. To determine the differential roles of serum and colony-stimulating factor (CSF) during the growth of FDC-P1 cultures, we investigated the kinetics of proliferation and death after withdrawal of serum or CSF, using flow cytometry to quantitate the numbers of vital and dead cells. After withdrawal of CSF, the cells died without entering a quiescent state. The life span of cultures lacking CSF increased with increasing concentrations of serum (greater than 50 h at 30% serum), and the cells kept dividing until they died. During the period of population death caused by the absence of CSF, the re-addition of CSF immediately prevented further cells from dying. After the withdrawal of serum in the presence of CSF, the cells continued to live and proliferate for weeks, but required high cell densities (much greater than 10(5)/ml), which suggests that the cells produced an active substance that can substitute for serum. Serum as well as serum-free conditioned medium from dense cultures made the survival and growth of FDC-P1 cultures independent of cell density. Without sufficient quantities of this activity, all cells of the population died within an interval that was much shorter than one cell cycle, which indicates that the factor acts throughout most of the cell cycle. The results suggest that both the CSF and the serum factor act together to permit cell survival, rather than to drive proliferation.     

In vitro analysis of cardiac progenitor cell differentiation

Cardiac myoblast commitment and differentiation were studied in the developing avian embryo. Single cell analysis of isolated cardiogenic cells grown in vitro established that stage 4 (newly gastrulated) mesodermal cells are capable of myocyte differentiation in the absence of intercellular contact or short range cellular interactions. While cardiac myocytes derived from single isolated progenitors expressed muscle-specific myosin heavy chains (MHC), atrial and ventricular MHCs characteristic of in vivo development were not detected. When the same progenitors were grown at high density or in organ cultures, cell-specific, expression of atrial and ventricular MHCs was observed, suggesting a role of cell density-dependent processes for differential MHC expression. Cardiogenic mesoderm (stages 4-8) was treated with the cocarcinogen 12-O-tetradecanoylphorbol-13-acetate (TPA), maintained as organ cultures, and assayed for muscle differentiation in an attempt to identify possible stage-specific variations in cardiac progenitors. TPA irreversibly blocked the differentiation of early (stages 4-7) progenitors. When exposed to TPA, stages 4-7 cardiogenic cells failed to synthesize several muscle-specific proteins as determined by immunochemical analysis of myosin synthesis and two-dimensional gel electrophoresis of 35S-labeled proteins isolated from cardiogenic cultures. In addition, stages 4-7, TPA-treated cells did not differentiate after the withdrawal of TPA. In contrast, TPA had no effect on the expression of several muscle-specific proteins in late (stage 8) cells including the cell-specific expression of atrial and ventricular MHCs.     

A computational model for cell differentiation

In this paper, we present a word set generating mechanism, called cell-differentiation system, inspired by the tissue process formation in multicellular organisms, which might model some properties of evolving communities of living cells at the syntactical level. The tools utilized to model these biological phenomena belong to the formal language theory. In this context chromosomal mutations are defined as operations on strings and the differentiation according to the control of gene expression is represented by some random-context conditions in formal languages.In the presented formal framework we prove that in a simplified form of this formalism, with only one cell-type which is regular, one single cell and no mitosis involved, the problem of establishing whether or not the set of vectors of integers indicating the number of cells in each population, is finite, linear or semilinear, is recursively undecidable. However, one can algorithmically decide whether or not a cell-differentiation system of finite cell-type can produce a specific generation of cells.     

Low concentration of ethanol favors progenitor cell differentiation and neovascularization in high-fat diet-fed mice model

Endothelial progenitor cells (EPCs) and monocytic cells from bone marrow (BM) can be recruited to the injured endothelium and contribute to its regeneration. During metabolic diseases such as obesity and diabetes, progenitor cell function is impaired. Several studies have shown that moderate alcohol consumption prevents the development and progression of atherosclerosis in a variety of animal/mouse models and increases mobilization of progenitor cells. Along with these studies, we identify ethanol at low concentration as therapeutic tool to in vitro expand progenitor cells in order to obtain an adequate number of cells for their use in the treatment of cardiovascular diseases.We evaluated the effects of ethanol on the phenotype of BM-derived cells from mice fed with high-fat diet (HFD). HFD did not induce changes in weight of mice but induced metabolic alterations. HFD feeding increased the differentiation of monocytic progenitors but not EPCs. Whereas ethanol at 0.6% is able to increase monocytic progenitor differentiation, 1% ethanol diminished it. Furthermore, ethanol at 0.6% increased the ability of progenitor cells to promote in vivo angiogenesis as well as secretome of BM-derived cells from mice fed with HFD, but not in mice fed normal diet. In conclusion, ethanol at low concentration is able to increase angiogenic abilities of progenitor cells from animals with early metabolic alterations.     

The network of hemopoietic regulatory proteins in myeloid cell differentiation.

There are clones of myeloid leukemic cells that can be induced to undergo terminal cell differentiation to macrophages by normal hemopoietic regulatory proteins. Induction of differentiation in two different clones of myeloid leukemic cells with interleukin 6 (IL-6) or granulocyte-macrophage colony-stimulating factor (GM-CSF) resulted in induction of mRNA for the hemopoietic regulatory proteins IL-6, GM-CSF, interleukin 1 alpha and interleukin 1 beta, tumor necrosis factor, and transforming growth factor beta 1. In one of these clones, induction of differentiation with GM-CSF was also associated with induction of mRNA for macrophage colony-stimulating factor (M-CSF) but not for the receptor for M-CSF (c-fms), whereas in the other clone, induction of differentiation with IL-6 was associated with induction of mRNA for both c-fms and M-CSF. The clones also differed in their responsiveness to these regulators. There was no induction of mRNA for granulocyte colony-stimulating factor or interleukin 3 during differentiation of either clone. The results indicate that the genes for a nearly normal network of positive and negative hemopoietic regulatory proteins are induced during differentiation of these myeloid leukemic cells and that there are leukemic clones with specific defects in this network.     

Evidence for distinct intracellular signaling pathways in CD34+ progenitor to dendritic cell differentiation from a human cell line model

Intracellular signals that mediate differentiation of pluripotent hemopoietic progenitors to dendritic cells (DC) are largely undefined. We have previously shown that protein kinase C (PKC) activation (with phorbol ester (PMA) alone) specifically induces differentiation of primary human CD34+ hemopoietic progenitor cells (HPC) to mature DC. We now find that cytokine-driven (granulocyte-macrophage CSF and TNF-alpha) CD34+ HPC-->DC differentiation is preferentially blocked by inhibitors of PKC activation. To further identify intracellular signals and downstream events important in CD34+ HPC-->DC differentiation we have characterized a human leukemic cell line model of this process. The CD34+ myelomonocytic cell line KG1 differentiates into dendritic-like cells in response to granulocyte-macrophage CSF plus TNF-alpha, or PMA (with or without the calcium ionophore ionomycin, or TNF-alpha), with different stimuli mediating different aspects of the process. Phenotypic DC characteristics of KG1 dendritic-like cells include morphology (loosely adherent cells with long neurite processes), MHC I+/MHC IIbright/CD83+/CD86+/CD14- surface Ag expression, and RelB and DC-CK1 gene expression. Functional DC characteristics include fluid phase macromolecule uptake (FITC-dextran) and activation of resting T cells. Comparison of KG1 to the PMA-unresponsive subline KG1a reveals differences in expression of TNF receptors 1 and 2; PKC isoforms alpha, beta I, beta II, and mu; and RelB, suggesting that these components/pathways are important for DC differentiation. Together, these findings demonstrate that cytokine or phorbol ester stimulation of KG1 is a model of human CD34+ HPC to DC differentiation and suggest that specific intracellular signaling pathways mediate specific events in DC lineage commitment.     

Homing of hemopoietic progenitor cells to the marrow

The recognition of hemopoietic stem cell after intravenous transplantation of marrow cells occurs initially by a lectin moiety on the surface of marrow sinus endothelium. The cell is then transported across the endothelial cytoplasm much in the way that a soluble ligand, such as transferrin, is transported. In the extravascular compartment, the cell binds to lineage-specific stromal cells. This mechanism, known as homing, is mediated by a lectin-glycoconjugate interaction, the lectin being on the surface of progenitor cell with specificity for galactosyl and mannosyl residues. The binding is subsequently stabilized by membrane-bound proteoglycans, integrin-like receptors, and fibronectin.     

Minor histocompatibility antigens on canine hemopoietic progenitor cells

Adoptive immunotherapy with CTL against minor histocompatibility Ags (mHA) provides a promising way to treat leukemia relapse in allogeneic chimeras. Here we describe the in vitro generation of CTL against mHA in the dog. We tested their inhibitory effect on the growth of hemopoietic progenitor cells stimulated by hemopoietic growth factors in a 4-day suspension culture. CTL were produced by coculture of donor PBMC with bone marrow-derived dendritic cells (DCs). These DCs were characterized by morphology, high expression of MHC class II and CD1a, and the absence of the monocyte-specific marker CD14. Characteristically these cells stimulated allogeneic lymphocytes (MLR) and, after pulsing with a foreign Ag (keyhole limpet hemocyanin), autologous T cells. CTL were generated either ex vivo by coculture with DCs of DLA-identical littermates or in vivo by immunization of the responder with DCs obtained from a DLA-identical littermate. In suspension culture assays the growth of hemopoietic progenitor cells was inhibited in 53% of DLA-identical littermate combinations. In canine families mHA segregated with DLA as restriction elements. One-way reactivity against mHA was found in five littermate combinations. In two cases mHA might be Y chromosome associated, in three cases autosomally inherited alleles were detected. We conclude that CTL can be produced in vitro and in vivo against mHA on canine hemopoietic progenitor cells using bone marrow-derived DCs.