Maureen Lynch

Fate tracing reveals the endothelial origin of hematopoietic stem cells

Hematopoietic stem cells (HSCs) originate within the aortic-gonado-mesonephros (AGM) region of the midgestation embryo, but the cell type responsible for their emergence is unknown since critical hematopoietic factors are expressed in both the AGM endothelium and its underlying mesenchyme. Here we employ a temporally restricted genetic tracing strategy to selectively label the endothelium, and separately its underlying mesenchyme, during AGM development. Lineage tracing endothelium, via an inducible VE-cadherin Cre line, reveals that the endothelium is capable of HSC emergence. The endothelial progeny migrate to the fetal liver, and later to the bone marrow, and are capable of expansion, self-renewal, and multilineage hematopoietic differentiation. HSC capacity is exclusively endothelial, as ex vivo analyses demonstrate lack of VE-cadherin Cre induction in circulating and fetal liver hematopoietic populations. Moreover, AGM mesenchyme, as selectively traced via a myocardin Cre line, is incapable of hematopoiesis. Our genetic tracing strategy therefore reveals an endothelial origin of HSCs.

The Notch/Jagged Pathway Inhibits Proliferation of Human Hematopoietic Progenitors In Vitro

The cell surface receptor Notch1 is expressed on CD34+ hematopoietic precursors, whereas one of its ligands, Jagged1, is expressed on bone marrow stromal cells. To examine the role of Notch signaling in early hematopoiesis, human CD34+ cells were cultured in the presence or absence of exogenous cytokines on feeder layers that either did or did not express Jagged1. In the absence of recombinant growth factors, Jagged1 decreased myeloid colony formation by CD34+ cells, as well as 3H‐thymidine incorporation and entry into S phase. In the presence of a strong cytokine signal to proliferate and mature, (interleukin 3 [IL‐3] and IL‐6, stem cell factor [SCF], and G‐CSF), Jagged1 did not significantly alter either the fold expansion or the types of colonies formed by CD34+ cells. However, in the presence of SCF alone, Jagged1 increased erythroid colony formation twofold. These results demonstrate that Notch can modulate a growth factor signal, and that in the absence of growth factor stimulation, the Jagged1‐Notch pathway preserves CD34+ cells in an immature state.

Vascular remodeling of the vitelline artery initiates extravascular emergence of hematopoietic clusters

The vitelline artery is a temporary structure that undergoes extensive remodeling during midgestation to eventually become the superior mesenteric artery (also called the cranial mesenteric artery, in the mouse). Here we show that, during this remodeling process, large clusters of hematopoietic progenitors emerge via extravascular budding and form structures that resemble previously described mesenteric blood islands. We demonstrate through fate mapping of vascular endothelium that these mesenteric blood islands are derived from the endothelium of the vitelline artery. We further show that the vitelline arterial endothelium and subsequent blood island structures originate from a lateral plate mesodermal population. Lineage tracing of the lateral plate mesoderm demonstrates contribution to all hemogenic vascular beds in the embryo, and eventually, all hematopoietic cells in the adult. The intraembryonic hematopoietic cell clusters contain viable, proliferative cells that exhibit hematopoietic stem cell markers and are able to further differentiate into myeloid and erythroid lineages. Vitelline artery-derived hematopoietic progenitor clusters appear between embryonic day 10 and embryonic day 10.75 in the caudal half of the midgut mesentery, but by embryonic day 11.0 are sporadically found on the cranial side of the midgut, thus suggesting possible extravascular migration aided by midgut rotation.

Identification of Novel Functional Regions Important for the Activity of HOXB7 in Mammalian Cells

Members of the HOX family of homeobox transcription factors play a role in pattern formation in diverse developmental systems. The clearly documented role of HOX genes in the proliferation and differentiation of primary hematopoietic cells and cell lines provides a convenient system to pursue a biochemical analysis of HOX gene function in mammalian cells. To explore the role of HOXB7 in myeloid hematopoiesis, a number of mutations and deletions in the gene were constructed that targeted sequences with known functions or in regions that had not been examined previously. The wild-type and mutant B7 constructs were introduced into the murine myelomonocytic cell line, 32D, and assayed for their effects on G-CSF-induced myeloid differentiation. Wild-type HOXB7 inhibited the differentiation of 32D cells, whereas mutations in the Pbx-binding pentapeptide motif or the DNA-binding homeodomain, as well as internal deletions of the N-terminal unique region, blocked this effect. Interestingly, mutations eliminating two target sites for casein kinase II, the glutamate-rich C terminus, or the first 14 amino acids of HOXB7, led to enhanced 32D differentiation. A model proposing a role for these regions of HOXB7 is presented.

Conjugation of an anti–transferrin receptor IgG3-avidin fusion protein with biotinylated saporin results in significant enhancement of its cytotoxicity against malignant hematopoietic cells

We have previously developed an antibody fusion protein composed of a mouse/human chimeric IgG3 specific for the human transferrin receptor genetically fused to avidin (anti-hTfR IgG3-Av) as a universal delivery system for cancer therapy. This fusion protein efficiently delivers biotinylated FITC into cancer cells via TfR-mediated endocytosis. In addition, anti-hTfR IgG3-Av alone exhibits intrinsic cytotoxic activity and interferes with hTfR recycling, leading to the rapid degradation of the TfR and lethal iron deprivation in certain malignant B-cell lines. We now report on the cytotoxic effects of a conjugate composed of anti-hTfR IgG3-Av and biotinylated saporin 6 (b-SO6), a toxin derived from the plant Saponaria officinalis that inhibits protein synthesis. Conjugation of anti-hTfR IgG3-Av with b-SO6 enhances the cytotoxic effect of the fusion protein in sensitive cells and also overcomes the resistance of malignant cells that show low sensitivity to the fusion protein alone. Our results show for the first time that loading anti-hTfR IgG3-Av with a biotinylated toxin enhances the cytotoxicity of the fusion protein alone. These results suggest that anti-hTfR IgG3-Av has great potential as a therapeutic agent for a wide range of applications due to its intrinsic cytotoxic activity plus its ability to deliver biotinylated molecules into cancer cells. [Mol Cancer Ther 2007;6(11):2995–3008]

The homeobox gene Hhex regulates the earliest stages of definitive hematopoiesis

The development and emergence of the hematopoietic stem cell involves a series of tightly regulated molecular events that are not well characterized. The hematopoietically expressed homeobox (Hhex) gene, a member of the homeobox gene family, is an essential regulator of embryogenesis and hematopoietic progenitor development. To investigate the role of Hhex in hematopoiesis we adapted a murine embryonic stem (ES) cell coculture system, in which ES cells can differentiate into CD41(+) and CD45(+) hematopoietic progenitors in vitro. Our results show that in addition to delayed hemangioblast development, Hhex(-/-) ES-derived progeny accumulate as CD41(+) and CD41(+)c-kit(+) cells, or the earliest definitive hematopoietic progenitors. In addition, Hhex(-/-) ES-derived progeny display a significantly reduced ability to develop into mature CD45(+) hematopoietic cells. The observed reduction in hematopoietic maturation was accompanied by reduced proliferation, because Hhex(-/-) CD41(+)CD45(-)c-kit(+) hematopoietic progenitors accumulated in the G(2) phase of the cell cycle. Thus, Hhex is a critical regulator of hematopoietic development and is necessary for the maturation and proliferation of the earliest definitive hematopoietic progenitors.

Regulation of expression of genes for enzymes of the mammalian urea cycle in permanent cell-culture lines of hepatic and non-hepatic origin

SummaryWe present here the results of investigations conducted by ourselves and others on the regulation of the expression of genes encoding the enzymes of the mammalian urea cycle as manifest in cultured cells of both hepatic and extrahepatic origin. Upon consideration of the recently discovered discrete non-hepatic arginase genetic locus in man and our consequent hypothesis that the form of arginase thus transcribed in such extrahepathic cells functions principally in providing ornithine for protein anabolism and polyamine biosynthesis, rather than in detoxifying ammonia through urea formation, we have chosen instead to study permanent cell lines that are derived from liver and continue to perform a variety of hepatic functions in culture as experimental models for probing the molecular mechanisms underlying the control of ureagenesis within the mature liver cell. Of two such arginase-positive rat-hepatoma lines, we have characterized extensively in one (H4-11-E-C3) the mode of action of glucocorticoids in augmenting the cellular levels of this enzyme as well as of argininosuccinate synthetase. To this end, we have recently demonstrated that these stimulations are both mediated by binding of the hormones to classical cytoplasmic steroid receptors in a specific and saturable fashion and have thus concluded that the H4-11-E-C3 line will provide a suitable cell culture system for subsequent more detailed experiments from which the information garnered will continue to be relevant to the ureagenic pathway as modulated in the differentiated hepatocyte in vivo.

Modeling Notch Signaling in Normal and Neoplastic Hematopoiesis: Global Gene Expression Profiling in Response to Activated Notch Expression

In normal hematopoiesis, proliferation is tightly linked to differentiation in ways that involve cell‐cell interaction with stromal elements in the bone marrow stem cell niche. Numerous in vitro and in vivo studies strongly support a role for Notch signaling in the regulation of stem cell renewal and hematopoiesis. Not surprisingly, mutations in the Notch gene have been linked to a number of types of malignancies. To better define the function of Notch in both normal and neoplastic hematopoiesis, a tetracycline‐inducible system regulating expression of a ligand‐independent, constitutively active form of Notch1 was introduced into murine E14Tg2a embryonic stem cells. During coculture, OP9 stromal cells induce the embryonic stem cells to differentiate first to hemangioblasts and subsequently to hematopoietic stem cells. Our studies indicate that activation of Notch signaling in flk+ hemangioblasts dramatically reduces their survival and proliferative capacity and lowers the levels of hematopoietic stem cell markers CD34 and c‐Kit and the myeloid marker CD11b. Global gene expression profiling of day 8 hematopoietic progenitors in the absence and presence of activated Notch yield candidate genes required for normal hematopoietic differentiation, as well as putative downstream targets of oncogenic forms of Notch including the noncanonical Wnts Wnt4 and 5A.

Regulation by glucocorticoids of rat-liver phenylalanine hydroxylase In Vivo

Phenylalanine hydroxylase, a liver-associated enzyme, is induced markedly by glucocorticoids in two permanent rat-hepatoma cell lines. In order to gain evidence that this phenomenon also occurs in vivo, we examined the effect of adrenalectomy and/or hormone supplementation on the levels of phenylalanine hydroxylase in the livers of adult rats: glucocorticoid administration increases, and adrenal ablation reduces, the activity of the hepatic enzyme, and the diminution occurring in the latter instance is entirely prevented by concurrent hormone replacement. These results thus corroborate earlier findings from a single experiment and are consistent with the hypothesis that adrenal corticosteroid hormones participate in modulating phenylalanine-hydroxylase levels within the diploid hepatocyte.

Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny

The in vitro differentiation of ES cells towards a hematopoietic cell fate is useful when studying cell populations that are difficult to access in vivo and for characterizing the earliest genes involved in hematopoiesis, without having to deal with embryonic lethalities. The ES/OP9 co-culture system was originally designed to produce hematopoietic progeny, without the over production of macrophages, as the OP9 stromal cell line is derived from the calvaria of osteopetrosis mutant mice that lack functional M-CSF. The in vitro ES/OP9 co-culture system can be used in order to recapitulate early hematopoietic development. When cultured on OP9 stromal cells, ES cells differentiate into Flk-1+ hemangioblasts, hematopoietic progenitors, and finally mature, terminally differentiated lineages. The standard ES/OP9 co-culture protocol entails the placement of ES cells onto a confluent layer of OP9 cells; as well as, periodic replating steps in order to remove old, contaminating OP9 cells. Furthermore, current protocols involve evaluating only the hematopoietic cells found in suspension and are not optimized for evaluation of ES-derived progeny at each day of differentiation. However, with replating steps and the harvesting of only suspension cells one potentially misses a large portion of ES-derived progeny and developing hematopoietic cells. This issue becomes important to address when trying to characterize hematopoietic defects associated with knockout ES lines. Here we describe a modified ES/mStrawberry OP9 co-culture, which allows for the elimination of contaminating OP9 cells from downstream assays. This method allows for the complete evaluation of all ES-derived progeny at all days of co-culture, resulting in a hematopoietic differentiation pattern, which more directly corresponds to the hematopoietic differentiation pattern observed within the embryo.