Erythroid progenitor cells and stimulating factors during murine embryonic and fetal development

Murine embryonic and fetal yolk-sacs, peripheral blood, and livers were assayed for hemopoietic multipotential and progenitor cell content between days 6 and 13 of gestation. Multipotential cells (Mix-CFC), erythroid-committed progenitor cells (BFU-E), and nonerythroid progenitor cells (predominantly GM-CFC) were assayed by their ability to form hemopoietic colonies in vitro when stimulated by pokeweed-mitogen-stimulated spleen-cell-conditioned media (as a source of Multi-CSF) and either human or murine erythropoietin. Late erythroid progenitor cells (CFU-E) were stimulated to form colonies by erythropoietin. Mix-CFC, BFU-E, and nonerythroid cells were first detected on day 8 in yolk-sacs, day 9 in peripheral blood, and day 11 in liver. Maximum absolute numbers of yolk-sac Mix-CFC (182), BFU-E (331), and non-erythroid CFC (1358) occurred at 11 days of gestation. The maximum frequency of peripheral blood mix-CFC (24/10(5) cells) and BFU-E (55/10(5) cells) occurred at ten days of gestation. The absolute numbers of hepatic Mix-CFC, BFU-E, nonerythroid CFC, and CFU-E increased exponentially from 11 to 13 days' gestation. CFU-E were first detected at nine days in peripheral blood, at ten days in yolk-sac, and 11 days in liver and at all ages were equally responsive to erythropoietin. The maximum frequency (151/10(5) cells) of CFU-E in the peripheral blood and the maximum number per yolk-sac (1699) both occurred on day 11 of gestation. In confirmation of previous studies, yolk-sac fluid was found to contain a macrophage colony-stimulating activity. In addition, an activity capable of stimulating fetal liver CFU-E was also detected in yolk-sac fluid. However, no activity (Multi-CSF) capable of stimulating Mix-CFC or BFU-E was detected in either yolk-sac fluid or fetal plasma.     

Specification of multipotential cardiovascular progenitor cells during embryonic stem cell differentiation and embryonic development

The fully formed heart is composed of diverse cell lineages including myocytes, endothelial cells, vascular smooth muscle cells, and fibroblasts that derive from distinct subsets of mesoderm during embryonic development. Findings from lineage tracing studies indicate that cardiomyocytes develop from cells that express fetal liver kinase-1, suggesting that the cardiac lineages may arise from a progenitor cell with vascular cardiomyocyte potential. Recent studies using the embryonic stem cell model have led to the identification of a fetal liver kinase-1(+) progenitor cell that displays both vascular and cardiomyocyte potential. A comparable progenitor was also isolated from the early mouse embryo. Identification and isolation of these cardiovascular progenitor cells establishes a new model of heart development that will provide insights into the mechanisms regulating cardiovascular lineage diversification. These progenitor cells may also represent a novel cell population for models of congenital heart disease and cell replacement therapy.     

Adult hemoglobins are synthesized in erythroid colonies in vitro derived from murine circulating hemopoietic progenitor cells during embryonic development.

Circulating peripheral blood cells and disaggregated yolk sac cells were obtained from normal mouse embryos as early as day 9 of gestation, prior to the formation of the fetal liver. These were cultured in vitro in plasma clots or methylcellulose, in the presence of either embryonic fluid or adult spleen cell-conditioned medium, with or without added erythropoietin. Large erythroid colonies were observed by the sixth day of culture. In all instances, these erythroid colonies synthesized adult hemoglobins. These results indicate that erythroid progenitor cells committed to adult hemoglobin synthesis are present in early embryonic circulation.     

Restricted spatiotemporal expression of lactoferrin during murine embryonic development

Lactoferrin is a member of the transferrin family of iron-binding glycoproteins. Lactoferrin is induced by estrogen in the mouse uterus during early pregnancy. However, the expression and function, if any, of lactoferrin in the preimplantation embryo during this developmental period has not been investigated. In the current study, the spatiotemporal expression of lactoferrin during murine embryogenesis was examined using in situ hybridization and immunohistochemical analyses. Lactoferrin expression was first detected in the 2-4 cell fertilized embryo and continued until the blastocyst stage of development. Interestingly, at the 16-cell stage, coinciding with the first major differentiation step in the embryo, lactoferrin messenger RNA (mRNA) is synthesized by the inner cells, whereas the protein is selectively taken up by the outside cells. This differential pattern of lactoferrin messenger RNA and protein localization continues until the blastocyst stage, with expression almost absent in the hatched blastocyst. Lactoferrin expression does not resume in the embryo until the latter half of gestation, where it is first detected in neutrophils of the fetal liver at embryonic day 11.5 and later in epithelial cells of the respiratory and digestive systems. Our results show that lactoferrin is expressed in a tightly regulated spatiotemporal manner during murine embryogenesis and suggest a novel paracrine role for this protein in the development of the trophoectodermal lineage during preimplantation development.     

Hemoglobin switching in culture: evidence for a humoral factor that induces switching in adult and neonatal but not fetal erythroid cells.

An erythropoietic activity that exerts a profound effect on fetal Hb synthesis is present in fetal sheep sera and it attains a peak concentration at the end of the second to the middle of the third trimester of fetal life. The activity consistently inhibits the increased synthesis of fetal Hb in cultures of burst-forming units (BFUes) from normal adults. In cultures of BFUes from homozygous beta+-thalassemias the activity produces a striking decline in gamma chain synthesis, a decline in G gamma/A gamma chain synthesis ratio, and an increase in delta/gamma and alpha/non-alpha ratios--i.e., findings suggesting a genuine gamma-to-beta switch. The activity accelerates Hb F-to-Hb A switching in neonatal BFUe cultures but it has no effect on fetal Hb synthesis in cultures of BFUe obtained from human fetuses. These findings provide direct evidence that (a) humoral factors play a role in the regulation of the switch from fetal to adult Hb formation, and (b) progenitor cells from various stages of ontogeny respond differently to these factors. The results are compatible with the hypothesis that Hb switching during development is mediated through a change in a developmental program which controls the responsiveness of progenitor cells to "switching" activities in their environment.     

Maternal serotonin is crucial for murine embryonic development

The early appearance of serotonin and its receptors during prenatal development, together with the many effects serotonin exerts during CNS morphogenesis, strongly suggest that serotonin influences the development and maturation of the mammalian brain before it becomes a neuromodulator/neurotransmitter. Sites of early serotonin biosynthesis, however, have not been detected in mouse embryos or extraembryonic structures, suggesting that the main source of serotonin could be of maternal origin. This hypothesis was tested by using knockout mice lacking the tph1 gene, which is responsible for the synthesis of peripheral serotonin. Genetic crosses were performed to compare the phenotype of pups born from homozygous and heterozygous mothers. Observations provide the first clear evidence that (i) maternal serotonin is involved in the control of morphogenesis during developmental stages that precede the appearance of serotonergic neurons and (ii) serotonin is critical for normal murine development. Most strikingly, the phenotype of tph1-/- embryos depends more on the maternal genotype than on that of the concepti. Consideration of the maternal genotype may thus help to clarify the influence of other genes in complex diseases, such as mental illness.     

Effect of human plasmalipoproteins on erythropoietic progenitor cells in serum-free cultures

Summary The purpose of the present study was to investigate the influence of human lipoproteins on CFU-e and BFU-e proliferation from human bone marrow in a serum-free system. In our previously described miniaturized agar system the main lipoprotein-density-classes from human plasma, namely very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density lipoproteins (LDL), high density lipoproteins₂ (HDL₂) and HDL₃ and a mixture of all the five lipoproteins were added in rising concentrations (from 1/10 to normal human plasma concentration) to serum-free medium containing delipidated and deionized bovine serum albumin (BSA), iron saturated transferrin and erythropoietin. The results demonstrate that all lipoproteins markedly increased the CFU-e and BFU-e proliferation after 7 and 14 days of incubation, respectively. Moreover, the lipoproteins induced a shift towards a lower threshold concentration of erythropoietin. Serumlike conditions were obtained if LDL and the mixture of lipoproteins were added to serum-free medium. Furthermore, in the serum-free cultures a maturation to the mature erythrocyte could be found.     

Adherent platelets recruit and induce differentiation of murine embryonic endothelial progenitor cells to mature endothelial cells in vitro

The homing and differentiation mechanisms of endothelial progenitor cells (EPCs) at sites of vascular lesions are unclear. To investigate whether platelets play a role in the recruitment and differentiation of EPCs, we made use of a robust mouse embryonic EPC (eEPC) line that reliably differentiates to a mature endothelial phenotype. We found that platelets stimulate chemotaxis and migration of these murine eEPCs. Further, the substantial adhesion of murine eEPCs on immobilized platelets that occurs under dynamic flow conditions is inhibited by neutralizing anti-P-selectin glycoprotein ligand-1 and anti-VLA-4 (beta1-integrin) monoclonal antibodies but not by anti-CD11b (aM-integrin; macrophage antigen-1). Coincubation of murine eEPCs with platelets for 5 days induced differentiation of EPCs to mature endothelial cells as verified by positive von Willebrand factor immunofluorescence and detection of Weibel Palade bodies through electron microscopy. We conclude that platelets may play a critical part in the capture and subsequent differentiation of murine eEPCs at sites of vascular lesions, revealing a possible new role of platelets in neoendothelization after vascular injury.     

Essential role for Galpha13 in endothelial cells during embryonic development

Toward identifying the roles of protease-activated receptor-1 (PAR1) and other G protein-coupled receptors important for vascular development, we investigated the role of Galpha13 in endothelial cells in the mouse embryo. LacZ inserted into Galpha13 exon 1 was highly expressed in endothelial cells at midgestation. Endothelial-specific Galpha13 knockout embryos died at embryonic days 9.5-11.5 and resembled the PAR1 knockout. Restoration of Galpha13 expression in endothelial cells by use of a Tie2 promoter-driven Galpha13 transgene rescued development of endothelial-specific Galpha13 knockout embryos as well the embryonic day 9.5 vascular phenotype in Galpha13 conventional knockouts; transgene-positive Galpha13-/- embryos developed for several days beyond their transgene-negative Galpha13-/- littermates and then manifested a previously uncharacterized phenotype that included intracranial bleeding and exencephaly. Taken together, our results suggest a critical role for Galpha13 in endothelial cells during vascular development, place Galpha13 as a candidate mediator of PAR1 signaling in this process, and reveal roles for Galpha13 in other cell types in the mammalian embryo.     

Azidothymidine-induced depression of murine hemopoietic progenitor cells

The toxicity of the antiviral drug 3'-azido-3'-deoxythymidine was studied in vivo on murine hemopoietic progenitor cells and peripheral blood cells. The drug induced a marked decrease of all tested populations, showing a severe toxicity on hemopoiesis.     

A liquid culture system for murine megakaryocyte progenitor cells

A liquid culture system is described for murine megakaryocyte progenitor cells (CFU-M) in the presence of pokeweed-mitogen-stimulated spleen-cell conditioned medium. There were dose-related responses between the number of CFU-M developed and the number of cells cultured and the dosage of conditioned medium in this liquid culture system. Murine CFU-M were abundantly cloned in this system an the plating efficiency was similar in comparison with that in a plasma clot system. The acetylcholinesterase-positive colonies (more than 4 acetylcholinesterase-positive cells) were clearly seen on day 3 of culture, and they reached a maximum (60.5 +/- 10.7/2 x 10(5) cells) on day 7 of culture. Ultrastructural analyses of megakaryocytic maturation in this system showed that a few megakaryocytes produced platelets that were released in the culture medium on day 5 of culture. This liquid culture system is suitable for the study of the dynamic process of the megakaryocyte-platelet system.     

Ontogeny of corticosterone-inducible growth hormone-secreting cells during chick embryonic development

We reported that corticosterone administration into the albumen of fertile chicken eggs on embryonic day (e) 11 induces an increase in the population of GH-secreting cells. The present study evaluated the ontogeny, dose response, localization, and persistence of the glucocorticoid-induced increase in the somatotroph population during chicken embryonic development. Corticosterone (0, 0.02, 0.2, and 2 microg in 300 microl saline) was injected into separate eggs on e9, e10, e11, and e12, and the population of GH-secreting cells was assessed 2 days later using reverse hemolytic plaque assays. Corticosterone treatment on e9 or e10 was unable to increase the population of GH-secreting cells on e11 or e12. In contrast, 0.2 and 2 microg of corticosterone on e11 increased the population of GH-secreting cells on e13 (P < 0.05, n = 3 experiments) to 8.2 +/- 0.6 and 6.4 +/- 0.5% of all cells, respectively, relative to controls (2.4 +/- 0.2%). For e14 embryos treated on e12, only the 2 microg dose increased the proportion of GH-secreting cells (6.4 +/- 0.6%) relative to controls (3.6 +/- 0.4%). In a second experiment, 0, 0.02, 0.2, 2, and 20 microg of corticosterone were injected on e0, e8, e9, e10, e11, and e12, and the population of GH-secreting cells was assessed on e13 in all groups. No dose of corticosterone was effective when given on e0, e8, e9, or e10. The 0.2 microg and 2 microg doses increased the population of GH-secreting cells (7.6 +/- 0.9% and 6.7 +/- 0.8%, respectively) relative to controls (2.3 +/- 0.4%) when injected on e11 (P < 0.05, n = 4 experiments). The 2-microg dose also increased GH cell abundance when injected on e12 (5.6 +/- 0.4%), relative to controls (2.7 +/- 0.5%). Treatment with 20 microg on e11 and e12 induced the greatest responses (10.3 +/- 1.1% and 8.7 +/- 0.9%, respectively). However, in subsequent experiments, administration of 20 microg on e11 resulted in embryonic death by e18. In a third set of experiments, two groups of eggs were injected either with 2 microg of corticosterone in saline or saline alone on e11, and the number of GH-secreting cells was estimated on e13, e16, e19, and the day of hatch (d1). The population of GH-secreting cells in corticosterone treated embryos was significantly higher than in saline treated embryos only on e13 (7.1 +/- 0.8% and 2.7 +/- 0.3%, respectively). No significant differences were observed on e16 (12.4 +/- 1.5% and 13.6 +/- 1.2%), e19 (19.0 +/- 1.0% and 18.2 +/- 1.7%) and d1 (23.8 +/- 2.1% and 25.1 +/- 1.8%) between corticosterone treated and control embryos, respectively. In a fourth set of experiments, whole mount in situ hybridization indicated that injection of corticosterone on e11 induced GH messenger RNA expression in the caudal part of the pituitary gland on e13, where somatotrophs are located normally later in development. We conclude that corticosterone administration in ovo can increase the population of GH-secreting cells in the caudal anterior pituitary only during a small window of development between e11 and e13 and that this premature increase of GH-secreting cells does not affect the percentage of GH-secreting cells later in development.     

Immunohistochemical evidence for hepatic progenitor cells in liver diseases

BACKGROUND/AIM: Proliferative bile ductular reactions occur in a variety of liver diseases in humans. It is a matter of debate whether such reactions result from progenitor cell proliferation with biliary and hepatocytic differentiation, versus biliary metaplasia of damaged hepatocytes. We investigated bile ductular reactions in liver diseases, paying particular attention to the presence of cells with intermediate (hepatocytic/biliary) features (oval-like cells). METHODS: Five specimens each were selected of submassive hepatic necrosis and cirrhosis due to hepatitis B, hepatitis C, autoimmune hepatitis, alcohol injury, primary biliary cirrhosis and primary sclerosing cholangitis. Immunohistochemical stains were performed for biliary markers (cytokeratins [CKs] 7 and 19), as well as hepatocytic markers (HepParl and alpha-fetoprotein[AFP]) in sequential sections. The degree of staining of each cell type (biliary, hepatocytic, intermediate) was graded semiquantitatively. RESULTS: Hepatocytes always stained diffusely for HepParl, occasionally for CK7, and rarely for CK19. Biliary cells were always diffusely positive for CK7 and CK19, and rarely for HepParl. Intermediate cells were identified in all cases and showed widespread staining for both HepParl and CK7, and less commonly for CK19. AFP was not expressed in any cell type. The morphologic and immunohistochemical features of bile ductular reactions were similar in the different diseases. CONCLUSIONS: Proliferating hepatic parenchymal cells with intermediate (hepatocytic/biliary) morphologic features and combined immunophenotype can be identified in a variety of acute and chronic liver diseases. The similarity of bile ductular reactions among chronic hepatitic, alcoholic and biliary diseases suggests that they result from proliferation of oval-like progenitor cells.     

Cytotoxicity as an indicator for transport mechanism: evidence that murine bone marrow progenitor cells lack a high-affinity leucine carrier that transports melphalan in murine L1210 leukemia cells

Cytotoxicity of melphalan to murine L1210 leukemia cells was reduced to a limiting maximum value of 50% by 2-aminobicyclo[2,2,1]heptane-2- carboxylic acid (BCH), indicating that cytotoxicity is partially dependent on drug uptake by system L. L-Leucine, but not alpha- aminoisobutyric acid (AIB), completely reduced the remaining 50% of drug cytotoxicity. These results contrast with those obtained with a sensitive host tissue, the bone marow progenitor cells of the white cell series (CFU-C), in that a high-affinity leucine transport system corresponding to system L was not identified.     

Efficient generation of retinal progenitor cells from human embryonic stem cells

The retina is subject to degenerative conditions, leading to blindness. Although retinal regeneration is robust in lower vertebrates, regeneration does not occur in the adult mammalian retina. Thus, we have developed efficient methods for deriving retinal neurons from human embryonic stem (hES) cells. Under appropriate culture conditions, up to 80% of the H1 line can be directed to the retinal progenitor fate, and express a gene expression profile similar to progenitors derived from human fetal retina. The hES cell-derived progenitors differentiate primarily into inner retinal neurons (ganglion and amacrine cells), with functional glutamate receptors. Upon coculture with retinas derived from a mouse model of retinal degeneration, the hES cell derived retinal progenitors integrate with the degenerated mouse retina and increase in their expression of photoreceptor-specific markers. These results demonstrate that human ES cells can be selectively directed to a neural retinal cell fate and thus may be useful in the treatment of retinal degenerations.     

Direct evidence for interaction between human erythroid progenitor cells and a hemoglobin switching activity present in fetal sheep serum.

An activity that induces Hb F to Hb A switching in human cells is present in fetal sheep serum. To test directly the role of cell-to-environment interactions in hemoglobin switching and to define the level of erythroid cell differentiation at which this activity operates, colony transfer experiments were done. Clones grown in the presence of switching activity-containing medium (fetal sheep serum) or control medium (fetal calf serum) were transferred, at the 16- to 30-cell stage, to either fetal sheep serum or fetal calf serum plates and Hb F synthesis was determined in the fully mature erythroid bursts. Fetal calf serum-to-fetal calf serum transfers produced colonies with the high Hb F levels characteristic of undisturbed fetal calf serum-grown clones. Fetal sheep serum-to-fetal calf serum transfers resulted in significant decrease in Hb F synthesis, revealing an interaction between hemoglobin switching activity and cells at an early stage of progenitor cell development. The reduction of Hb F synthesis in fetal calf serum-to-fetal sheep serum transfers indicated that hemoglobin switching activity interacts with cells at later stages of progenitor cell development. Maximal decrease in Hb F synthesis was observed in fetal sheep serum-to-fetal sheep serum transfers, indicating that optimal effects on Hb switching are obtained when the environment that induces Hb switching is present throughout the development of progenitor cells. By splitting single early clones into two parts and transferring them to either a fetal sheep serum or a fetal calf serum environment, these interactions were further demonstrated in the progeny of a single erythroid burst-forming unit. Since all clone transfers were done on cell-free plates, the results of fetal calf serum-to-fetal sheep serum and of fetal sheep serum-to-fetal sheep serum transfers indicated that the switching activity does not require helper cells for its action. These studies show directly that (i) Hb F synthesis is controlled at the level of progenitors and (ii) it involves interactions between progenitor cells and their environment.