Differentiated cell types derived from human embryonic stem cells (hESCs) may serve in the future to treat various human diseases. A crucial step toward their successful clinical application is to examine the immune response that might be launched against them after transplantation. We used two experimental platforms to examine the in vivo leukocyte response toward hESCs. First, immunocompetent and immunodeficient mouse strains were used to identify T cells as the major component that causes xenorejection of hESCs. Second, mice that were conditioned to carry peripheral blood leukocytes from human origin were used to test the human leukocyte alloresponse toward undifferentiated and differentiated hESCs. Using this model, we have detected only a minute immune response toward undifferentiated as well as differentiated hESCs over the course of 1 month, although control adult grafts were repeatedly infiltrated with lymphocytes and destroyed. Our data show that the cells evade immune destruction due to a low immunostimulatory potential. Nevertheless, a human cytotoxic T lymphocyte clone that was specifically prepared to recognize two hESC lines could lyse the cells after major histocompatibility complex class I (MHC-I) induction. Although MHC-I levels in hESCs are sufficient for rejection by cytotoxic T cells, our data suggest that the immunostimulatory capacity of the cells is very low. Thus, immunosuppressive regimens for hESC-based therapeutics could be highly reduced compared with conventional organ transplantation because direct allorejection processes of hESCs and their derivatives are considerably weaker. STEM CELLS 2006;24:221-229

The CX 3 C chemokine family is composed of only one member, CX 3 CL1, also known as fractalkine, which in mice is the sole ligand of the G proteincoupled, 7-transmembrane receptor CX 3 CR1. Unlike classic small peptide chemokines, CX 3 CL1 is synthesized as a membrane-anchored protein that can promote integrin-independent adhesion. Subsequent cleavage by metalloproteases, either constitutive or induced, can generate shed CX 3 CL1 entities that potentially have chemoattractive activity. To study the CX 3 C interface in tissues of live animals, we generated transgenic mice (CX 3 CL1 cherry :CX 3 CR1 gfp ), which express red and green fluorescent reporter genes under the respective control of the CX 3 CL1 and CX 3 CR1 promoters. Furthermore, we performed a structure/function analysis to differentiate the in vivo functions of membranetethered versus shed CX 3 CL1 moieties by comparing their respective ability to correct established defects in macrophage function and leukocyte survival in IntroductionChemokine (CX 3 C motif) ligand 1 (CX 3 CL1), also known as fractalkine or neurotactin, 1,2 and its receptor CX 3 CR1 3 have been assigned their own CX 3 C chemokine family. This classification is based on the 3 amino acid gap between its N-terminal cysteines in CX 3 CL1, with no spacing in CC chemokines and only one intervening amino acid in CXC chemokines. 4 CX 3 CL1 is furthermore structurally unique in that it is synthesized as a type I transmembrane protein with the CX 3 C chemokine domain presented on an extended stalk. 1,2 Both CX 3 CL1 and CX 3 CR1 are widely expressed throughout the organism; but in given tissues, expression is often highly cell type-specific. Taking advantage of mice that harbor a targeted replacement of the CX 3 CR1 gene by a GFP reporter, 5 we could, for instance, show that CX 3 CR1 expression in the brain is restricted to microglia. CX 3 CR1 expression in the gut was found limited to lamina propria macrophages and CX 3 CR1 expression in the blood is largely restricted to monocytes, which are uniform CX 3 CR1 positive, albeit with discrete expression levels. 6 CX 3 CR1 is furthermore expressed by macrophage/ dendritic cell precursors, 7 various dendritic cell (DC) progenitors, a nonclassic CD8␣ ϩ DC subset, 8 and plasmacytoid DCs. Aside from the prominent expression in the mononuclear myeloid compartment, CX 3 CR1 receptor expression has been reported for an NK cell subset 3,9 and certain T-cell populations. 3,10,11 The in vivo expression pattern of the ligand CX 3 CL1 remains less well defined and controversial 12 but has been reported for neurons, 13 intestinal epithelium, 14 and inflamed endothelium. 2 Notably, in humans eotaxin-3/CC chemokine ligand 26 was recently reported to be a functional ligand for CX 3 CR1 15 ; in mice, the CCL26 gene, however, is a pseudogene.The analysis of CX 3 C receptor and ligand knockout mice 5,16 has revealed a number of phenotypes resulting from the lack of CX 3 CR1/ CX 3 CL1 interactions. [17][18][19][20] However, in-depth knowledge of the physiologic rol...

Hematopoietic stem and progenitor cells (HSPCs) are regulated by various bone marrow stromal cell types. Here we identified rare activated bone marrow monocytes and macrophages with high expression of α-smooth muscle actin (α-SMA) and the cyclooxygenase COX-2 that were adjacent to primitive HSPCs. These myeloid cells resisted radiation-induced cell death and further upregulated COX-2 expression under stress conditions. COX-2-derived prostaglandin E(2) (PGE(2)) prevented HSPC exhaustion by limiting the production of reactive oxygen species (ROS) via inhibition of the kinase Akt and higher stromal-cell expression of the chemokine CXCL12, which is essential for stem-cell quiescence. Our study identifies a previously unknown subset of α-SMA(+) activated monocytes and macrophages that maintain HSPCs and protect them from exhaustion during alarm situations.

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Mechanisms governing stress-induced hematopoietic progenitor cell mobilization are not fully deciphered. We report that during granulocyte colony-stimulating factor-induced mobilization c-Met expression and signaling are up-regulated on immature bone marrow progenitors. Interestingly, stromal cell-derived factor 1/CXC chemokine receptor-4 signaling induced hepatocyte growth factor production and IntroductionDuring steady state conditions, adhesive interactions between the bone marrow (BM) stromal cells and primitive hematopoietic cells mostly result in stem cell retention, in a noncycling and nonmotile mode. However, low levels of progenitor cells are continuously released from the BM to the blood circulation as part of homeostasis. This process is dramatically amplified during emergency situations because of damage and cell death, as part of host defense and repair, in response to stress signals, including cytokines such as granulocyte colony-stimulating factor (G-CSF). Repetitive G-CSF stimulations are commonly used in the clinic, mimicking emergency situations to harvest stem and progenitor cells from the circulation for transplantation protocols. 1,2 The BM reservoir of immature and maturing leukocytes is dynamic, replenishing the blood with new cells on demand. These dynamic changes are achieved through a complex interplay between the immune and nervous systems, the bones and the BM microenvironment, involving cytokines, chemokines, proteolytic enzymes, and adhesion molecules. 3 In particular, oscillations in BM levels of stromal cell-derived factor 1 (SDF-1; transiently increased and subsequently degraded) and CXC chemokine receptor-4 (CXCR4) activation play a crucial role in promoting progenitor cell egress. 4,5 The cytokine hepatocyte growth factor (HGF) and its receptor c-Met control complex biologic programs known as "invasive growth" and tumor spreading. 6 Reactive oxygen species (ROS) are constantly generated during intracellular metabolism and in response to cytokines. Although excess ROS can cause oxidative damage to DNA, moderate levels have important roles in cell signaling, regulating different physiologic and pathologic cellular processes, including cell-cycle progression, migration, and invasion. 7 Finally, redox signaling has emerged as an important regulator of hematopoietic stem cell (HSC) self-renewal and lifespan. 8,9 The Forkhead Box, class O (FOXO) family of Forkhead transcription factors is a regulator of oxidative stress. 10 Loss of FOXO function in HSCs results in increased ROS levels, defective maintenance of quiescence, and reduced long-term repopulating ability. 11,12 FOXOs are a direct substrate of the protein kinase Akt, a mammalian target of rapamycin inhibition (mTOR) target, 13 which inactivates them by phosphorylation. 14 In this study, we demonstrate that c-Met expression levels on immature and maturing leukocytes in the BM reservoir are dynamic and dramatically increased when urgent requirements for enhanced leukocyte production and recruitment emerge. Moreover, full c-Me...

Repair of injured lungs represents a longstanding therapeutic challenge. We show that human and mouse embryonic lung tissue from the canalicular stage of development (20-22 weeks of gestation for humans, and embryonic day 15-16 (E15-E16) for mouse) are enriched with progenitors residing in distinct niches. On the basis of the marked analogy to progenitor niches in bone marrow (BM), we attempted strategies similar to BM transplantation, employing sublethal radiation to vacate lung progenitor niches and to reduce stem cell competition. Intravenous infusion of a single cell suspension of canalicular lung tissue from GFP-marked mice or human fetal donors into naphthalene-injured and irradiated syngeneic or SCID mice, respectively, induced marked long-term lung chimerism. Donor type structures or 'patches' contained epithelial, mesenchymal and endothelial cells. Transplantation of differentially labeled E16 mouse lung cells indicated that these patches were probably of clonal origin from the donor. Recipients of the single cell suspension transplant exhibited marked improvement in lung compliance and tissue damping reflecting the energy dissipation in the lung tissues. Our study provides proof of concept for lung reconstitution by canalicular-stage human lung cells after preconditioning of the pulmonary niche.

Xenotransplantation of pig tissues has great potential to overcome the shortage of organ donors. One approach to address the vigorous immune rejection associated with xenotransplants is the use of embryonic precursor tissue, which induces and utilizes host vasculature upon its growth and development. Recently, we showed in mice that embryonic pig pancreatic tissue from embryonic day 42 (E42) exhibits optimal properties as a ␤ cell replacement therapy. We now demonstrate the proof of concept in 2 diabetic Cynomolgus monkeys, followed for 393 and 280 days, respectively. A marked reduction of exogenous insulin requirement was noted by the fourth month after transplantation, reaching complete independence from exogenous insulin during the fifth month after transplantation, with full physiological control of blood glucose levels. The porcine origin of insulin was documented by a radioimmunoassay specific for porcine C-peptide. Furthermore, the growing tissue was found to be predominantly vascularized with host blood vessels, thereby evading hyperacute or acute rejection, which could potentially be mediated by preexisting anti-pig antibodies. Durable graft protection was achieved, and most of the late complications could be attributed to the immunosuppressive protocol. While fine tuning of immune suppression, tissue dose, and implantation techniques are still required, our results demonstrate that porcine E-42 embryonic pancreatic tissue can normalize blood glucose levels in primates. Its long-term proliferative capacity, its revascularization by host endothelium, and its reduced immunogenicity, strongly suggest that this approach could offer an attractive replacement therapy for diabetes.immune-suppression ͉ rejection ͉ xeno-transplantation

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