Lymphohematopoietic progenitors do not have a synchronized defect with age-related thymic involution

It has been speculated that aging lymphohematopoietic progenitor cells (LPC) including hematopoietic stem cells (HSC) and early T-cell progenitors (ETP) have intrinsic defects that trigger age-related thymic involution. However, using a different approach, we suggest that that is not the case. We provided a young thymic microenvironment to aged mice by transplanting a fetal thymus into the kidney capsule of aged animals, and demonstrated that old mouse-derived LPCs could re-establish normal thymic lymphopoiesis and all thymocyte subpopulations, including ETPs, double negative subsets, double positive, and CD4(+) and CD8(+) single positive T cells. LPCs derived from aged mice could turn over young RAG(-/-) thymic architecture by interactions, as well as elevate percentage of peripheral CD4(+)IL-2(+) T cells in response to costimulator in aged mice. Conversely, intrathymic injection of ETPs sorted from young animals into old mice did not restore normal thymic lymphopoiesis, implying that a shortage and/or defect of ETPs in aged thymus do not account for age-related thymic involution. Together, our findings suggest that the underlying cause of age-related thymic involution results primarily from changes in the thymic microenvironment, causing extrinsic, rather than intrinsic, defects in T-lymphocyte progenitors.     

Purification and functional assay of pluripotent hematopoietic stem cells

Hematolymphopoietic stem cells (HSC) have the capacity for extensive self-renewal and pluripotent myelolymphoid differentiation. Recent studies have emphasized the heterogeneity of human HSC subsets in terms of proliferative and self-renewal capacity. In the NOD-SCID (nonobese diabetic-severe combined immunodeficient) mouse xenograft assay, most CD34+38- stem cell clones proliferate at early times, but then disappear, whereas only few clones persist: possibly, the latter ones consist of long-term engrafting CD34+38- HSC expressing the KDR receptor (i.e. the vascular endothelial growth factor receptor II). In this regard, isolation of the small KDR+ subset from the CD34+ hematopoietic progenitors (and possibly from the CD34-lin- population) may provide a novel and effective approach for the purification of long-term proliferating HSC. More importantly, KDR+ HSC isolation will pave the way to cellular/molecular characterization and improved functional manipulation of HSC/HSC subsets, as well as to innovative approaches for HSC clinical utilization, specifically transplantation, transfusion medicine and gene therapy.     

Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood

Mouse hematopoiesis is initiated by long-term hematopoietic stem cells (HSC) that differentiate into a series of multipotent progenitors that exhibit progressively diminished self-renewal ability. In human hematopoiesis, populations enriched for HSC activity have been identified, as have downstream lineage-committed progenitors, but multipotent progenitor activity has not been uniquely isolated. Previous reports indicate that human HSC are enriched in Lin-CD34+CD38- cord blood and bone marrow and express CD90. We demonstrate that the Lin-CD34+CD38- fraction of cord blood and bone marrow can be subdivided into three subpopulations: CD90+CD45RA-, CD90-CD45RA-, and CD90-CD45RA+. Utilizing in vivo transplantation studies and complementary in vitro assays, we demonstrate that the Lin-CD34+CD38-CD90+CD45RA- cord blood fraction contains HSC and isolate this activity to as few as 10 purified cells. Furthermore, we report the first prospective isolation of a population of candidate human multipotent progenitors, Lin-CD34+CD38-CD90-CD45RA- cord blood.     

The direct profibrotic and indirect immune antifibrotic balance of blocking the cannabinoid 2 receptor

Cannabinoid 2 (CB2) receptors expressed on immune cells are considered to be antifibrogenic. Hepatic stellate cells (HSCs) directly interact with phagocytosis lymphocytes, but the nature of this interaction is obscure. We aimed to study the effects of CB2 receptors on hepatic fibrosis via their role in mediating immunity. Hepatic fibrosis was induced by carbon-tetrachloride (CCl(4)) administration in C57BL/6 wild-type (WT) and CB2 knockout (CB2(-/-)) mice. Irradiated animals were reconstituted with WT or CB2(-/-) lymphocytes. Lymphocytes from na?ve/fibrotic WT animals and healthy/cirrhotic hepatitis C virus were preincubated in vitro with or without CB2 antagonist, evaluated for proliferation and apoptosis, and then cocultured with primary mouse HSCs or a human HSC line (LX2), respectively. Lymphocyte phagocytosis was then evaluated. Following CCl(4)-administration, CB2(-/-) mice developed significant hepatic fibrosis but less necroinflammation. WT mice harbored decreased liver CD4(+) and NK(+) cells but increased CD8(+) subsets. Na?ve CB2(-/-) mice had significantly decreased T cell subsets. Adoptive transfer of CB2(-/-) lymphocytes led to decreased fibrosis in the irradiated WT recipient compared with animals receiving WT lymphocytes. Moreover, necroinflammation also tended to decrease. In vitro, a CB2-antagonist directly increased human HSC activation and increased apoptosis and decreased proliferation of mice/human T cells (healthy/fibrotic) and their phagocytosis. We concluded that CB2(-/-) lymphocytes exert an antifibrotic activity, whereas lack of CB2 receptor in HSCs promotes fibrosis. These findings broaden our understanding of cannabinoid signaling in hepatic fibrosis beyond their activity solely in HSCs.     

SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells

To improve our ability to identify hematopoietic stem cells (HSCs) and their localization in vivo, we compared the gene expression profiles of highly purified HSCs and non-self-renewing multipotent hematopoietic progenitors (MPPs). Cell surface receptors of the SLAM family, including CD150, CD244, and CD48, were differentially expressed among functionally distinct progenitors. HSCs were highly purified as CD150(+)CD244(-)CD48(-) cells while MPPs were CD244(+)CD150(-)CD48(-) and most restricted progenitors were CD48(+)CD244(+)CD150(-). The primitiveness of hematopoietic progenitors could thus be predicted based on the combination of SLAM family members they expressed. This is the first family of receptors whose combinatorial expression precisely distinguishes stem and progenitor cells. The ability to purify HSCs based on a simple combination of SLAM receptors allowed us to identify HSCs in tissue sections. Many HSCs were associated with sinusoidal endothelium in spleen and bone marrow, though some HSCs were associated with endosteum. HSCs thus occupy multiple niches, including sinusoidal endothelium in diverse tissues.     

Deceptive multilineage reconstitution analysis of mice transplanted with hemopoietic stem cells, and implications for assessment of stem cell numbers and lineage potentials

Hemopoietic stem cells (HSC) are identified through their unique ability, at the single cell level, to long-term reconstitute all blood cell lineages. Sustained myeloid reconstitution is considered the hallmark of HSC, because myeloid progenitors and their progeny have very short half-lives. Here we demonstrate that the established practice of relying on RB6-8C5 as a myeloid specific Ab can result in overestimation of HSC frequencies because the RB6-8C5 Ab also detects Ags expressed on a sizeable population of CD3(+)CD8(+) T cells, constitutively as well as following transplantation. Likewise, a high fraction of mice transplanted with limiting numbers of ex vivo expanded Lin(-)Sca(+)kit(+)CD34(-) HSC show long-term RB6-8C5(+)CD3(+) (lymphoid) but no RB6-8C5(+)CD3(-) (myeloid) reconstitution. Most noteworthy, the use of RB6-8C5 as a myeloid specific Ab can be deceptive by implicating the existence of lineage-restricted HSC capable of long-term reconstituting the myeloid and T, but not B, cell lineage. Because cross-lineage expression of "lineage-specific" markers is unlikely to be unique to the blood system, claims of unexpected cell fates should be substantiated not only by acquisition of lineage-specific markers, but also absence of markers of other lineages normally derived from the investigated stem cells.     

Effects of murine endotoxemia on lymphocyte subsets and clearance of staphylococcal pulmonary infection

In a model of staphylococcal pneumonia initiated during systemic endotoxemia in BALB/c mice, a significant reduction of the number of circulating CD4+ and CD8+ T-lymphocytes, B-lymphocytes, and NK cells, as well as lung-resident total T- and CD4+ T-lymphocytes was demonstrated. Staphylococcus aureus exposure only induced a similar decrease of lymphocyte subsets in the blood. However, the number of lung-resident total T- and CD4+ T-lymphocytes was increased. More viable bacteria were recovered from the lungs of S. aureus-infected mice than from those animals previously treated with lipopolysaccharide (LPS) followed by a staphylococcal challenge. These results indicate that LPS-induced reduction in the number of circulating lymphocyte subsets and lung-resident total T- and CD4+ T-lymphocytes do not increase susceptibility to staphylococcal respiratory infection. Moreover, LPS challenge prior to S. aureus exposure significantly improves clearance of the bacteria in the lung.     

Intrahepatic transplantation of CD34+ cord blood stem cells into newborn and adult NOD/SCID mice induce differential organ engraftment

In vivo studies concerning the function of human hematopoietic stem cells (HSC) are limited by relatively low levels of engraftment and the failure of the engrafted HSC preparations to differentiate into functional immune cells after systemic application. In the present paper we describe the effect of intrahepatically transplanted CD34⁺ cells from cord blood into the liver of newborn or adult NOD/SCID mice on organ engraftment and differentiation.Analyzing the short and long term time dependency of human cell recruitment into mouse organs after cell transplantation in the liver of newborn and adult NOD/SCID mice by RT-PCR and FACS analysis, a significantly high engraftment was found after transplantation into liver of newborn NOD/SCID mice compared to adult mice, with the highest level of 35% human cells in bone marrow and 4.9% human cells in spleen at day 70. These human cells showed CD19 B-cell, CD34 and CD38 hematopoietic and CD33 myeloid cell differentiation, but lacked any T-cell differentiation. HSC transplantation into liver of adult NOD/SCID mice resulted in minor recruitment of human cells from mouse liver to other mouse organs. The results indicate the usefulness of the intrahepatic application route into the liver of newborn NOD/SCID mice for the investigation of hematopoietic differentiation potential of CD34⁺ cord blood stem cell preparations.     

Prevention of endotoxin-induced lethality, but not of liver apoptosis in poly(ADP-ribose) polymerase-deficient mice.

Activation of poly-(ADP-ribose) polymerase (PARP) is often associated with cytotoxicity, but its precise role in shock-induced lethality and in different modes of tissue injury is still unknown. We took advantage of the existence of mice with a targeted deletion of the PARP gene (PARP-/-) to examine the differential sensitivity of wild-type (wt) and PARP-/- mice toward endotoxin (LPS)-induced lethality and different forms of liver damage. All PARP-/- animals survived high-dose (20 mg/kg) LPS-mediated shock, which killed 60% of wt animals. Moreover, LPS-induced necrotic liver damage was significantly reduced. In contrast, when apoptotic liver damage was induced via injection of low concentrations of LPS (30 microgram/kg) into D-galactosamine-sensitized mice, or via activation of hepatic cell death receptors, PARP-/- animals were not protected. We conclude that PARP is involved in systemic LPS toxicity, while it plays a minor role in apoptotic liver damage mediated by TNF or CD95.     

Requirement for CD44 in homing and engraftment of BCR-ABL-expressing leukemic stem cells

In individuals with chronic myeloid leukemia (CML) treated by autologous hematopoietic stem cell (HSC) transplantation, malignant progenitors in the graft contribute to leukemic relapse, but the mechanisms of homing and engraftment of leukemic CML stem cells are unknown. Here we show that CD44 expression is increased on mouse stem-progenitor cells expressing BCR-ABL and that CD44 contributes functional E-selectin ligands. In a mouse retroviral transplantation model of CML, BCR-ABL1-transduced progenitors from CD44-mutant donors are defective in homing to recipient marrow, resulting in decreased engraftment and impaired induction of CML-like myeloproliferative disease. By contrast, CD44-deficient stem cells transduced with empty retrovirus engraft as efficiently as do wild-type HSCs. CD44 is dispensable for induction of acute B-lymphoblastic leukemia by BCR-ABL, indicating that CD44 is specifically required on leukemic cells that initiate CML. The requirement for donor CD44 is bypassed by direct intrafemoral injection of BCR-ABL1-transduced CD44-deficient stem cells or by coexpression of human CD44. Antibody to CD44 attenuates induction of CML-like leukemia in recipients. These results show that BCR-ABL-expressing leukemic stem cells depend to a greater extent on CD44 for homing and engraftment than do normal HSCs, and argue that CD44 blockade may be beneficial in autologous transplantation in CML.     

Targeting human CD34+ hematopoietic stem cells with anti-CD45 x anti-myosin light-chain bispecific antibody preserves cardiac function in myocardial infarction.

We have previously shown that targeting human CD34(+) hematopoietic stem cells (HSC) with a bispecific antibody (BiAb) directed against myosin light chain (MLC) increases delivery of cells to the injured hearts and improves cardiac performance in the nude rat. In this study, we have sought to validate our previous observations and to perform more detailed determination of ventricular function in immunocompetent mice with myocardial infarction (MI) that were treated with armed CD34(+) HSC. We examined whether armed CD34(+) HSC would target the injured heart following MI and restore ventricular function in vitro. MI was created by ligation of the left anterior descending artery. After 48 h, adult ICR mice received either 0.5 x 10(6) human CD34(+) HSC armed with anti-CD45 x anti-MLC BiAb or an equal volume of medium through a single tail vein injection. Two weeks after stem cell administration, ventricular function of hearts from mice receiving armed CD34(+) HSC was significantly greater compared with the same parameters from control mice. Immunohistochemistry confirmed the accumulation of CD34(+) HSC in MI hearts infused with stem cells. Angiogenesis was significantly enhanced in CD34(+) HSC-treated heart as determined by vascular density per area. Furthermore, histopathological examination revealed that the retained cardiac function observed in CD34(+) HSC-treated mice was associated with decreased ventricular fibrosis. These results suggest that peripheral administration of armed CD34(+) HSC results in localization of CD34(+) HSC to injured myocardium and restores myocardial function.     

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer''s patch (PP). Common dendritic cell progenitors (CDPs, lin^- c-kit^lo CD115⁺ Flt3⁺) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar^-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1⁺ and recipients being CD45.2⁺. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar^-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.     

Hematopoietic stem cell aging is associated with functional decline and delayed cell cycle progression

The molecular mechanisms underlying hematopoietic stem cell (HSC) aging remain to be elucidated. In this study, we investigated age-related changes in the functional and phenotypic properties of murine HSCs. Consistent with previous studies, we found that the number and frequency of CD34^−/lowc-Kit⁺Sca-1⁺lineage marker⁻ (CD34⁻KSL) cells, a highly enriched HSC population, significantly increased in old mice, though their repopulating ability was reduced. Continuous bromodeoxyuridine labeling revealed a significant delay in the cell cycle progression of CD34⁻KSL cells in old mice. This delay was also observed in young recipients transplanted with whole bone marrow cells from old mice. When cultured in vitro, CD34⁻KSL cells from old mice showed a greater capacity to give rise to primitive CD48⁻KSL cells with reduced HSC activity. Gene expression profiling identified age-related changes in the expression of several cell cycle regulatory genes, including p21/Cdkn1a and p18/Cdkn2c. These results support the notion that HSC aging is largely regulated by an intrinsic genetic program.     

Stem cell factor consistently improves thymopoiesis after experimental transplantation of murine or human hematopoietic stem cells in immunodeficient mice

Deficient thymopoiesis is a pivotal determinant of impaired immune competence following hematopoietic stem cell transplantation (HSCT). Stem cell factor (SCF) is essentially involved in early thymopoiesis. We evaluated whether SCF administration would improve recovery of thymopoiesis following HSCT in immunodeficient mice receiving: 1) bone marrow (BM) transplantation of congenic mice; or 2) human fetal liver HSCT in the human immune system mouse model. Following murine BM transplantation, SCF significantly enhanced thymopoiesis and peripheral T cell recovery in lymph nodes and spleen. SCF did not affect BM lymphoid progenitor recovery and/or expansion. Median thymic cellularity increased from 0.9 in PBS- to 266 × 10(4)/thymus in SCF-treated mice (p = 0.05). Following human HSCT in human immune system mice, higher thymic cellularity was observed in SCF-treated mice. Double-negative and early double-positive thymocyte subsets increased, but especially late double-positive, CD4 single-positive, and CD8 single-positive thymocyte subsets were significantly enhanced (p < 0.05). These results show that exogenous supply of SCF may significantly improve murine and human posttransplant thymopoiesis, for which the effect is probably exerted by directly promoting T cell development intrathymically rather than by enhanced entry of prethymically expanded lymphoid progenitors.     

NK cells play a critical role in the regulation of class I-deficient hemopoietic stem cell engraftment: evidence for NK tolerance correlates with receptor editing

The role that NK cells play in the rejection of hemopoietic stem cell (HSC) and tolerance induction has remained controversial. In this study, we examined whether NK cells play a direct role in the rejection of HSC. Purified HSC from MHC class II-deficient mice engrafted readily in congenic mice, while HSC from class I-deficient donors (beta(2)-microglobulin(-/-) (beta(2)m(-/-))) failed to engraft. Recipient mice lacking CD8(+), CD4(+), or T cells also rejected HSC from class I-deficient donors, pointing directly to NK cells as the effector in rejection of HSC. Recipients, deficient in or depleted of NK cells, engrafted readily with beta(2)m(-/-) HSC. Expression of the activating Ly-49D and inhibitory Ly-49G2 receptors on recipient NK cells was significantly decreased in these beta(2)m(-/-)-->B6 chimeras, and the proportion of donor NK cells expressing Ly-49D was also significantly decreased. Notably, beta(2)m(-/-) chimeras accepted beta(2)m(-/-) HSC in second transplants, demonstrating that NK cells in the chimeras had been tolerized to beta(2)m(-/-). Taken together, our data demonstrate that NK cells play a direct role in the regulation of HSC engraftment, and down-regulation and/or deletion of specific NK subsets in mixed chimeras can contribute to the induction of NK cell tolerance in vivo. Moreover, our data show that bone marrow-derived elements significantly contribute to NK cell development and tolerance.