Detailed cytological analysis of cells undergoing differentiation often reveals clues to the regulation of multiple cell features. The Hedgehog (Hh) signaling cascade is a master regulator of cell fate during differentiation and is implicated in the development of some neoplasias. Hh signaling affects the expression of cell surface markers of differentiation. We have used the flow cytometer to evaluate the effect of blockage of the Hh signal on the expression of cell surface markers of erythroid differentiation in an in vitro system. In addition, the effect of Hh signaling on the distribution of cells in the phases of the cell cycle over the course of erythroid differentiation was assessed. Inhibition of the Hh signal retards progression of the erythroid developmental program. Included is a discussion of some of the basic parameters, limitations, and interpretations of flow cytometric analysis used for CD marker expression and cell cycle studies.

Tissue restoration is the process whereby multiple damaged cell types are replaced to restore the histoarchitecture and function to the tissue. Several theories, have been proposed to explain the phenomenon of tissue restoration in amphibians and in animals belonging to higher order. These theories include dedifferentiation of damaged tissues, transdifferentiation of lineage-committed progenitor cells, and activation of reserve, precursor cells. Studies by Young et al. and others demonstrated that connective tissue compartments throughout postnatal individuals contain reserve precursor cells. Subsequent repetitive single cell-cloning and cell-sorting studies revealed that these reserve precursor cells consisted of multiple populations of cells, including, tissue-specific progenitor cells, germ-layer lineage stem cells, and pluripotent stem cells. Tissue-specific progenitor cells display various capacities for differentiation, ranging from unipotency (forming a single cell type) to multipotency (forming multiple cell types). However, all progenitor cells demonstrate a finite life span of 50 to 70 population doublings before programmed cell senescence and cell death occurs. Germ-layer lineage stem cells can form a wider range of cell types than a progenitor cell. An individual germ-layer lineage stem cell can form all cells types within its respective germ-layer lineage (i.e., ectoderm, mesoderm, or endoderm). Pluripotent stem cells can form a wider range of cell types than a single germ-layer lineage stem cell. A single pluripotent stem cell can form cells belonging to all three germ layer lineages. Both germ-layer lineage stem cells and pluripotent stem cells exhibit extended capabilities for self-renewal, far surpassing the limited life span of progenitor cells (50–70 population doublings). The authors propose that the activation of quiescent tissue-specific progenitor cells, germ-layer lineage stem cells, and/or pluripotent stem cells may be a potential explanation, along with dedifferentiation and transdifferentiation, for the process of tissue restoration. Several model systems are currently being investigated to determine the possibilities of using these adult quiescent reserve precursor cells for tissue engineering.

Undifferentiated cells have been identified in the prenatal blastocyst, inner cell mass, and gonadal ridges of rodents and primates, including humans. After isolation these cells express molecular and immunological markers for embryonic cells, capabilities for extended self-renewal, and telomerase activity. When allowed to differentiate, embryonic stem cells express phenotypic markers for tissues of ectodermal, mesodermal, and endodermal origin. When implanted in vivo, undifferentiated noninduced embryonic stem cells formed teratomas. In this report we describe a cell clone isolated from postnatal rat skeletal muscle and derived by repetitive single-cell clonogenic analysis. In the undifferentiated state it consists of very small cells having a high ratio of nucleus to cytoplasm. The clone expresses molecular and immunological markers for embryonic stem cells. It exhibits telomerase activity, which is consistent with its extended capability for self-renewal. When induced to differentiate, it expressed phenotypic markers for tissues of ectodermal, mesodermal, and endodermal origin. The clone was designated as a postnatal pluripotent epiblastic-like stem cell (PPELSC). The undifferentiated clone was transfected with a genomic marker and assayed for alterations in stem cell characteristics. No alterations were noted. The labeled clone, when implanted into heart after injury, incorporated into myocardial tissues undergoing repair. The labeled clone was subjected to directed lineage induction in vitro, resulting in the formation of islet-like structures (ILSs) that secreted insulin in response to a glucose challenge. This study suggests that embryonic-like stem cells are retained within postnatal mammals and have the potential for use in gene therapy and tissue engineering.

Hedgehog (Hh) signaling regulates differentiation in numerous systems, but its functions in the control of hematopoietic differentiation have not been extensively explored. Initial studies have indicated that hedgehog signaling affects the proliferation and differentiation of erythroid progenitors (Detmer, K., et al., Erythroid differentiation in vitro is blocked by cyclopamine, an inhibitor of hedgehog signaling. Blood Cells Mol. Dis. 26(4) (2000) 360-372). To examine the effect of Hh signaling on the erythroid developmental program at the molecular level, Hh signaling in committed erythroid progenitors differentiating in vitro was inhibited, and the appearance/disappearance of molecular markers of erythroid differentiation was monitored. The expression timetable for CD34, CD36, the erythropoietin receptor, and glycophorin A was retarded in the absence of Hh signaling. Hemoglobinization was delayed and decreased relative to controls. Morphological changes of erythroid maturation were also delayed. The fraction of cells in S-phase was decreased during the initial period of exponential expansion as assessed by propidium iodide staining and flow cytometry, as was the rate of tritiated thymidine incorporation. A modest decrease in the proliferation rate was observed. These results suggest that Hh signaling is one of the mechanisms in the regulation of erythroid proliferation and differentiation.

BACKGROUND:Cytokines such as tumor necrosis factor alpha (TNF-alpha) are produced by the myocardium in heart disease and might be stimulated by reactive oxygen. In some cell types, cyclic adenosine monophosphate (AMP) inhibits TNF-alpha production. The authors tested the hypothesis that stimulation of cardiac beta-adrenergic receptors would inhibit cytokine gene transcription induced by reactive oxygen.METHODS:Rat hearts were perfused with buffer containing hypoxanthine. Reactive oxygen intermediates were generated by infusion of xanthine oxidase. Myocardial mRNA encoding 11 cytokines was determined. TNF-alpha, interleukin-6, and cyclic AMP were measured in the coronary effluent.RESULTS:In control hearts, of the screened RNA, only mRNA encoding interleukin-1beta, -4, and -6 was detected. Stimulation with hypoxanthine-xanthine oxidase (HX-XO) induced detectable mRNA for TNF-alpha and interleukin-5 and increased mRNA band density for interleukin-1beta, -4, and -6. Simultaneous infusion of isoproterenol inhibited HX-XO-stimulated cytokine gene expression and caused release of cyclic AMP into the coronary effluent. In control hearts, TNF-alpha was not detected in the coronary effluent. After HX-XO administration, TNF-alpha was reliably detected at 60 min and interleukin-6 at 90 min. Simultaneous infusion of isoproterenol inhibited TNF-alpha and interleukin-6 release. Inclusion of propranolol in the perfusion buffer blocked the isoproterenol-induced inhibition of HX-XO-stimulated TNF-alpha release and release of cyclic AMP into the coronary effluent. In addition, elevating myocardial cyclic AMP with forskolin also blocked release of TNF-alpha stimulated by HX-XO. Finally, delaying infusion of isoproterenol until 30 min after HX-XO administration still suppressed release of TNF-alpha.CONCLUSIONS:Reactive oxygen species activate cytokine gene transcription in the myocardium. The sympathetic nervous system, acting through beta-receptors to elevate myocardial cyclic AMP, regulates cardiac cytokine production by inhibition of transcription.

We examined the effects of bone morphogenetic protein-2 (BMP-2), -3, -4, -5, -6, and -7 on the proliferation and differentiation of bone marrow CD34+ haematopoietic progenitors in semi-solid medium. The BMPs had no effect on haematopoietic colony development when added to medium containing erythropoietin (Epo) or Interleukin-3 plus Epo. Synergistic effects with the haematopoietic cytokines stem cell factor (SCF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were observed. In conjunction with GM-CSF and Epo, BMP-4 increased the number of both erythroid and granulocyte/monocyte colonies formed in semi-solid medium (P<0.01). No other BMP stimulated erythroid colony development under these conditions, while BMP-3, BMP-7 (P<0.01), BMP-5, and BMP-6 (P<0.05) stimulated granulocyte/monocyte colony formation. BMP-7 acted synergistically with stem cell factor to increase granulocyte/monocyte colony formation but not erythroid colony formation. The other BMPs did not affect either erythroid or granulocyte/monocyte colony development under these conditions. These results suggest that individual BMPs form part of the complement of cytokines regulating the development of haematopoietic progenitors, and in particular, point to a role for BMP-4 in the control of definitive, as well as embryonic erythropoiesis.

BACKGROUND:Inflammatory reaction and intimal proliferation of smooth muscle cells are characteristics of vascular stenotic lesions. Nuclear factor kappaB (NF-kappaB) is involved in regulation of inflammation and cell survival in a variety of cell types. We tested a hypothesis that selective inhibition of NF-kappaB by expression of a mutated, nondegradable inhibitor of NF-kappaB, IkappaB-alphaM, would inhibit proinflammatory cytokine expression and proliferation in human vascular smooth muscle cell.MATERIALS AND METHODS:Smooth muscle cells were cultured from internal mammary artery and infected with recombinant adenovirus vectors.RESULTS:Adenoviral expression of IkappaB-alphaM inhibited diverse signal-triggered cellular IkappaB-alpha degradation, subsequent NF-kappaB activation, and transactivation of proinflammatory cytokine genes. Expression of IkappaB-alphaM in low-density VSMC led to a 60% reduction in serum-stimulated cell growth and a 10% increment in apoptotic incidence but was without effect in high-density cultures. Coexpression of NF-kappaB p65 attenuated apoptosis in low-density cells induced by IkappaB-alphaM. Therefore, the susceptibility to apoptosis induction in the low-density cells correlated with lower constitutive NF-kappaB activity. The induction of apoptosis by IkappaB-alphaM and the rescue by NF-kappaB p65 might be explained by mutual control of NF-kappaB p65 and IkappaB-alphaM access to the nucleus.CONCLUSION:Our results suggest that expression of nondegradable IkappaB-alpha might have therapeutic potential in both vascular inflammatory reaction and smooth muscle cell proliferation.

Because inflammatory processes may promote the development of atherosclerosis, we examined the activation of cytokine genes in rat vascular smooth muscle cells in vitro after treatment with bacterial lipopolysaccharide (LPS). Interleukin-1 (IL-1), IL-6 and tumor necrosis factor-alpha (TNF-alpha) mRNA increased in response to LPS. Activation of nuclear factor-kappaB (NF-kappaB) presumably results in NF-kappaB binding to regulatory regions of target genes and activating transcription. We therefore compared the kinetics of NF-kappaB activation, cytokine message production, and TNF-alpha secretion. Maximum active NF-kappaB was found at 30 min after the addition of LPS and decreased thereafter. Increased IL-6 mRNA was detected at 30 min, increased TNF-alpha mRNA at 60 min, and increased IL-1 mRNA at 120 min. Secretion of TNF-alpha was dependent on LPS concentration and was first detected 120 min after LPS addition. Aspirin, which has been shown to inhibit NF-kappaB activation and cytokine secretion in other cell types, did not inhibit NF-kappaB activation or TNF-alpha secretion. However, aspirin reduced the amount of both TNF-alpha and IL-6 mRNA present 30 min after LPS addition by half (P < 0.05).

Adult hematopoietic differentiation is a developmental process that employs many of the same molecular mechanisms as embryogenesis. To explore the possibility that hedgehog signaling is involved in the control of hematopoietic differentiation, we screened a panel of human leukemia cell lines for the expression of Patched1 and Smoothened, the receptor and coreceptor for hedgehog ligands. Expression was found in multiple cell lines, and Patched1 expression was detected in normal marrow. Induction of myeloid differentiation in cell lines downregulated expression of both genes. When normal marrow mononuclear cells were grown in semisolid medium in the presence of 10 microM cyclopamine, development of colonies of granulocytic/monocytic lineage was unaffected in terms of both number and morphology. The number of erythroid colonies, however, was significantly reduced (P < 0.01). Furthermore, hemoglobinization was substantially delayed relative to controls in those erythroid colonies that did form. Incubation of hematopoietic progenitors with Shh-N and GM-CSF resulted in increased granulocyte/monocyte colonies (P < 0.01); the increase was blocked by cyclopamine. Incubation of hematopoietic progenitors with Shh-N and stem cell factor resulted in larger erythroid colonies. These results suggest that elements of the hedgehog signaling pathway are involved in the control of hematopoietic differentiation.

Each year millions of people suffer tissue loss or end-stage organ failure. While allogeneic therapies have saved and improved countless lives, they remain imperfect solutions. These therapies are limited by critical donor shortages, long-term morbidity, and mortality. A wide variety of transplants, congenital malformations, elective surgeries, and genetic disorders have the potential for treatment with autologous stem cells as a source of HLA-matched donor tissue. Our current research is aimed at characterizing cell surface cluster differentiation (CD) markers on human progenitor and pluripotent cells to aid in isolating comparatively purified populations of these cells. This study examined human pluripotent and progenitor cells isolated from fetal, mature, and geriatric individuals for the possible presence of 15 CD markers. The response to insulin and dexamethasone revealed that the cell isolates were composed of lineage-committed progenitor cells and lineage-uncommitted pluripotent cells. Flow cytometry showed cell populations positive for CD10, CD13, CD56, and MHC Class-I markers and negative for CD3, CD5, CD7, CD11b, CD14, CD15, CD16, CD19, CD25, CD45, and CD65 markers. Northern analysis revealed that CD13 and CD56 were actively transcribed at time of cell harvest. We report the first identification of CD10, CD13, CD56, and MHC Class-I cell surface antigens on these human cells.

ABSTRACT: To explore the possibility that bone morphogenetic proteins (BMPs) are autocrine/paracrine regulators of hematopoietic differentiation and function, we screened a panel of human cell lines encompassing the hematopoietic lineages for expression of members of this family of genes. Expression of BMP-2, BMP-4, BMP-6, BMP-7, Growth and Differentiation Factor-1 (GDF-1), Placental Bone Morphogenetic Protein (PLAB), and Transforming Growth Factor-β3 (TGF-β3) was detected in one or more cell lines. BMP-2, BMP-4, BMP-7, and TGF-β3 expression was also found in normal hematopoietic tissue. Expression of BMP-5 and BMP-8 was not seen. Lineage-restricted patterns of expression were found for BMP-4 (T-lymphoid), BMP-7 (lymphoid), PLAB (macrophage/monocyte), and GDF-1 (myeloid). Expression of BMP-2, GDF-1, and PLAB could be modulated by treatment with differentiating agents. Marked variations in the levels of BMP-4, BMP-7, and PLAB expression were encountered, indicating that disorders in BMP signaling pathways may play a role in the development of hematopoietic neoplasia.

Alterations in alveolar macrophage (AM) function during sepsis-induced hypoxia may influence tumor necrosis factor (TNF) secretion and the progression of acute lung injury. Nuclear factor (NF)-κB is thought to regulate the expression of endotoxin [lipopolysaccharide (LPS)]-induced inflammatory cytokines such as TNF, and NF-κB may also be influenced by changes in O2tension. It is thus proposed that acute changes in O2tension surrounding AMs alter NF-κB activation and TNF secretion in these lung cells. AM-derived TNF secretion and NF-κB expression were determined after acute hypoxic exposure of isolated Sprague-Dawley rat AMs. Adhered AMs (106/ml) were incubated (37°C at 5% CO2) for 2 h with LPS ( Pseudomonas aeruginosa, 1 μg/ml) in normoxia (21% O2-5% CO2) or hypoxia (1.8% O2-5% CO2). AM-derived TNF activity was measured with a TNF-specific cytotoxicity assay. Electrophoretic mobility shift and supershift assays were used to determine NF-κB activation and to identify NF-κB isoforms in AM extracts. In addition, mRNAs for selected AM proteins were determined with RNase protection assays. LPS-exposed AMs in hypoxia had higher levels of TNF ( P < 0.05) and enhanced expression of NF-κB ( P < 0.05); the predominant isoforms were p65 and c-Rel. Increased mRNA bands for TNF-α, interleukin-1α, and interleukin-1β were also observed in the hypoxic AMs. These results suggest that acute hypoxia in the lung may induce enhanced NF-κB activation in AMs, which may result in increased production and release of inflammatory cytokines such as TNF.

Society of Critical Care Medicine; 27th Educational and Scientific Symposium; San Antonio, Texas, USA; February 4-8, 1998: Oral Presentations: Organ Injury I: Role of Intracellular Signalling Molecules, Saturday, February 7, 1998 8: 25-9: 40 am