Mineo Iwata

The Human Homolog of Rat Jagged1Expressed by Marrow Stroma Inhibits Differentiation of 32D Cells through Interaction with Notch1

A cDNA clone encoding the human homolog of rat Jagged1 was isolated from normal human marrow. Analyses of human stromal cell lines indicate that this gene, designated hJagged1, is expressed by marrow stromal cells typified by the cell line HS-27a, which supports the long-term maintenance of hematopoietic progenitor cells. G-CSF-induced differentiation of 32D cells expressing Notch1 was inhibited by coculturing with HS-27a. A peptide corresponding to the Delta/Serrate/LAG-2 domain of hJagged1 and supernatants from COS cells expressing a soluble form of the extracellular portion of hJagged1 were able to mimic this effect. These observations suggest that hJagged1 may function as a ligand for Notch1 and play a role in mediating cell fate decisions during hematopoiesis.

Progenitor cells identified by PDGFR-alpha expression in the developing and diseased human heart.

Platelet-derived growth factors (PDGFs) and their tyrosine kinase receptors play instrumental roles in embryonic organogenesis and diseases of adult organs. In particular, platelet-derived growth factor receptor-alpha (PDGFRα) is expressed by multipotent cardiovascular progenitors in mouse and human embryonic stem cell systems. Although cardiac PDGFRα expression has been studied in multiple species, little is known about its expression in the human heart. Using immunofluorescence, we analyzed PDGFRα expression in both human fetal and diseased adult hearts, finding strong expression in the interstitial cells of the epicardium, myocardium, and endocardium, as well as the coronary smooth muscle. Only rare endothelial cells and cardiomyocytes expressed PDGFRα. This pattern was consistent for both the fetal and adult diseased hearts, although more PDGFRα+ cardiomyocytes were noted in the latter. In vitro differentiation assays were then performed on the PDGFRα+ cell fraction isolated from the cardiomyocyte-depleted human fetal hearts. Protocols previously reported to direct differentiation to a cardiomyocyte (5-azacytidine), smooth muscle (PDGF-BB), or endothelial cell fates (vascular endothelial growth factor [VEGF]) were used. Although no significant cardiomyocyte differentiation was observed, PDGFRα+ cells generated significant numbers of smooth muscle cells (smooth muscle-α-actin+ and smooth muscle myosin+) and endothelial cells (CD31+). These data suggest that a subfraction of the cardiac PDGFRα+ populations are progenitors contributing predominantly to the vascular and mesenchymal compartments of the human heart. It may be possible to control the fate of these progenitors to promote vascularization or limit fibrosis in the injured heart.

Dissecting the Marrow Microenvironment

Abstract: Cloned human stromal cell lines representing functionally distinct cellular components of the marrow microenvironment were generated to serve as tools for identifying gene products that regulate hematopoiesis. Oligonucleotide arrays, or “gene chips” were used to provide a comprehensive comparison of gene expression among the cell lines. One line, designated HS‐5, was found to secrete large amounts of cytokines, and conditioned media from this line was found to support the ex vivo expansion of both immature and mature progenitors. In contrast, a second line, designated HS‐27a, does not secrete known cytokines but does support cobblestone area formation by CD34+/38lo cells. HS‐27a, but not HS‐5, was also found to express hJagged1, a ligand for Notch1, which may function to influence cell fate decisions of hematopoietic precursors. Both cell lines are currently being used to identify other gene products that regulate hematopoiesis and to generate reagents that will allow more formal evaluation of the putative role of hJagged1 in hematopoietic cell fate decisions.

Comparison of Human Embryonic Stem Cell-Derived Cardiomyocytes, Cardiovascular Progenitors, and Bone Marrow Mononuclear Cells for Cardiac Repair

Summary Cardiomyocytes derived from human embryonic stem cells (hESC-CMs) can improve the contractility of injured hearts. We hypothesized that mesodermal cardiovascular progenitors (hESC-CVPs), capable of generating vascular cells in addition to cardiomyocytes, would provide superior repair by contributing to multiple components of myocardium. We performed a head-to-head comparison of hESC-CMs and hESC-CVPs and compared these with the most commonly used clinical cell type, human bone marrow mononuclear cells (hBM-MNCs). In a nude rat model of myocardial infarction, hESC-CMs and hESC-CVPs generated comparable grafts. Both similarly improved systolic function and ventricular dilation. Furthermore, only rare human vessels formed from hESC-CVPs. hBM-MNCs attenuated ventricular dilation and enhanced host vascularization without engrafting long-term or improving contractility. Thus, hESC-CMs and CVPs show similar efficacy for cardiac repair, and both are more efficient than hBM-MNCs. However, hESC-CVPs do not form larger grafts or more significant numbers of human vessels in the infarcted heart.

Cytomegalovirus mediated myelosuppression

Cytomegalovirus (CMV) has long been associated with myelosuppression. Evidence for this association has been provided from in vitro studies, statistical analysis of clinical studies, informative case reports, and murine models. Reports differ as to how CMV mediates myelosuppression. Some data indicate direct infection of hematopoietic progenitors and their progeny, others indicate that the supportive microenvironment is infected and thereby its supportive function compromised. In this report we review data suggesting that the severity of myelosuppression in patients is associated with particular CMV genotypes identified by variations in the glycoprotein B gene.

Interleukin-1-Mediated Inhibition of Cytomegalovirus Replication Is Due to Increased IFN-β Production

Previous studies have demonstrated that the intercellular spread of cytomegalovirus (CMV) is reduced in marrow stromal cells that either secrete interleukin-1 (IL-1) or are treated with exogenous IL-1. Here, we report that IL-1-treated marrow stromal cells and fibroblasts, when infected with CMV, produce decreased amounts of infectious progeny virus. CMV-infected cells treated with IL-1 contained more interferon-beta (IFN-beta) mRNA at 24 h postinfection compared with untreated, infected cells. IFN-beta protein secreted into fibroblast culture supernatants increased from 46 +/- 1 IU/ml in untreated, infected cells to 116 +/- 5 IU/ml in IL-1-treated infected cells. When IFN-beta activity was inhibited, using blocking antibodies to either the cytokine or the IFN-alpha/beta receptor, the addition of IL-1 no longer limited viral spread. Furthermore, viral spread in nonIL-1-treated cultures was inhibited by the addition of recombinant IFN-beta. These studies suggest that IL-1 functions to limit CMV spread by increasing the expression of IFN-beta, which in turn reduces production of infectious virus.

Functionally and Phenotypically Distinct Subpopulations of Marrow Stromal Cells Are Fibroblast in Origin and Induce Different Fates in Peripheral Blood Monocytes

Marrow stromal cells constitute a heterogeneous population of cells, typically isolated after expansion in culture. In vivo, stromal cells often exist in close proximity or in direct contact with monocyte-derived macrophages, yet their interaction with monocytes is largely unexplored. In this report, isolated CD146(+) and CD146(-) stromal cells, as well as immortalized cell lines representative of each (designated HS27a and HS5, respectively), were shown by global DNase I hypersensitive site mapping and principal coordinate analysis to have a lineage association with marrow fibroblasts. Gene expression profiles generated for the CD146(+) and CD146(-) cell lines indicate significant differences in their respective transcriptomes, which translates into differences in secreted factors. Consequently, the conditioned media (CM) from these two populations induce different fates in peripheral blood monocytes. Monocytes incubated in CD146(+) CM acquire a tissue macrophage phenotype, whereas monocytes incubated in CM from CD146(-) cells express markers associated with pre-dendritic cells. Importantly, when CD14(+) monocytes are cultured in contact with the CD146(+) cells, the combined cell populations, assayed as a unit, show increased levels of transcripts associated with organismal development and hematopoietic regulation. In contrast, the gene expression profile from cocultures of monocytes and CD146(-) cells does not differ from that obtained when monocytes are cultured with CD146(-) CM. These in vitro results show that the CD146(+) marrow stromal cells together with monocytes increase the expression of genes relevant to hematopoietic regulation. In vivo relevance of these data is suggested by immunohistochemistry of marrow biopsies showing juxtaposed CD146(+) cells and CD68(+) cells associated with these upregulated proteins.

CDCP1 identifies a CD146 negative subset of marrow fibroblasts involved with cytokine production.

In vitro expanded bone marrow stromal cells contain at least two populations of fibroblasts, a CD146/MCAM positive population, previously reported to be critical for establishing the stem cell niche and a CD146-negative population that expresses CUB domain-containing protein 1 (CDCP1)/CD318. Immunohistochemistry of marrow biopsies shows that clusters of CDCP1+ cells are present in discrete areas distinct from areas of fibroblasts expressing CD146. Using a stromal cell line, HS5, which approximates primary CDCP1+ stromal cells, we show that binding of an activating antibody against CDCP1 results in tyrosine-phosphorylation of CDCP1, paralleled by phosphorylation of Src Family Kinases (SFKs) Protein Kinase C delta (PKC-δ). When CDCP1 expression is knocked-down by siRNA, the expression and secretion of myelopoietic cytokines is increased. These data suggest CDCP1 expression can be used to identify a subset of marrow fibroblasts functionally distinct from CD146+ fibroblasts. Furthermore the CDCP1 protein may contribute to the defining function of these cells by regulating cytokine expression.

Late Infusion of Cloned Marrow Fibroblasts Stimulates Endogenous Recovery from Radiation-Induced Lung Injury

In the current study, we used a canine model of radiation-induced lung injury to test the effect of a single i.v. infusion of 10×106/kg of marrow fibroblasts on the progression of damage following 15 Gy exposure to the right lung. The fibroblasts, designated DS1 cells, are a cloned population of immortalized cells isolated from a primary culture of marrow stromal cells. DS1 cells were infused at week 5 post-irradiation when lung damage was evident by imaging with high-resolution computed tomography (CT). At 13 weeks post-irradiation we found that 4 out of 5 dogs receiving DS1 cells had significantly improved pulmonary function compared to 0 out of 5 control dogs (p = 0.047, Fisher’s Exact). Pulmonary function was measured as the single breath diffusion capacity-hematocrit (DLCO-Hct), the total inspiratory capacity (IC), and the total lung capacity (TLC), which differed significantly between control and DS1-treated dogs; p = 0.002, p = 0.005, and p = 0.004, respectively. The DS1-treated dogs also had less pneumonitis detected by CT imaging and an increased number of TTF-1 (thyroid transcription factor 1, NKX2-1) positive cells in the bronchioli and alveoli compared to control dogs. Endothelial-like progenitor cells (ELC) of host origin, detected by colony assays, were found in peripheral blood after DS1 cell infusion. ELC numbers peaked one day after infusion, and were not detectable by 7 days. These data suggest that infusion of marrow fibroblasts stimulates mobilization of ELC, which is associated with a reduction in otherwise progressive radiation-induced lung injury. We hypothesize that these two observations are related, specifically that circulating ELC contribute to increased angiogenesis, which facilitates endogenous lung repair.

Stromal Inhibition of Myeloid Differentiation: A Possible Role for hJagged1

Cloned human stromal cell lines, HS-27a and HS-23, were established in our laboratories as previously described.1 HS-27a, unlike HS-23, supports the formation of cobblestone areas from immature CD34+/38− cells. Recently we reported that HS27a expresses a human homolog for rat Jagged1 (hJagged1), a ligand for Notch1, which may play a role in cell-fate decisions that prevent differentiation and allow cobblestone area formation.2 The purpose of the current study was to develop a model for investigating whether hJagged1 can prevent myeloid differentiation. Towards this end we used U937 cells, which can be induced to differentiate by exposure to all trans retinoic acid (ATRA). This model was attractive because the induction of differentiation could be easily followed by the expression of CD11b on the surface of the differentiating cells. Critical to the model was the observation that U937 cells do express considerable amounts of mRNA for Notch1 (data not shown), suggesting that the receptor may be present on these cells. Based on the observations that Notch/Jagged interactions induce several genes that have the recombination binding protein Jκ (RBPJκ) consensus sequence, we first used electrophoretic mobility shift assays (EMSA) to detect the induction of nuclear protein binding to this consensus sequence in U937 cells before and after they were cultured on either hJagged1-expressing HS-27a cells or hJagged1-negative HS23 cells. The data shown in FIGURE 1 indicate that the EMSA assay is specific for the DNA binding protein and, further, that nuclear protein binding to the RBPJκ consensus sequence was increased when Notch-expressing cells were cultured with HS27a, but not after coculturing with HS-23. This strongly suggested that Notch/Jagged signaling occurs in the U937 cells, although it is possible that some other, as yet undefined, molecule on HS-27a cells is responsible for inducing the increase in DNA binding protein. We next hypothesized that if Notch/Jagged signaling prevented U937 from differentiating in response to ATRA, this block would result in the failure of U937 cells to express CD11b, which is uniformly induced during differentiation. To test this hypothesis, U937 cells were cultured in ATRA alone or in the presence of HS-23 or HS-27a cells. After 4 days the nonadherent and adherent U937 cells were harvested separately and analyzed by fluorescence-activated cell sorter (FACS) for expression of CD11b. FIGURE 2 shows that U937 cells exposed to ATRA alone or in the pres-