Differential kinetics of primitive hematopoietic cells assayed in vitro and in vivo during serum-free suspension culture of CD34+ blood progenitor cells.

So far, blood progenitor cells (BPC) expanded ex vivo in the absence of stromal cells have not been demonstrated to reconstitute hematopoiesis in myeloablated patients. To characterize the fate of early hematopoietic progenitor cells during ex vivo expansion in suspension culture, human CD34(+)-enriched BPC were cultured in serum-free medium in the presence of FLT3 ligand (FL), stem cell factor (SCF) and interleukin 3 (IL-3). Both CD34 surface expression levels and the percentage of CD34+ cells were continuously downregulated during the culture period. We observed an expansion of colony-forming units granulocyte-macrophage (CFU-GM) and BFU-E beginning on day 3 of culture, reaching an approximate 2-log increase by days 5 to 7. Limiting dilution analysis of primitive in vitro clonogenic progenitors was performed through a week 6 cobblestone-area-forming cell (CAFC) assay, which has previously been shown to detect long-term bone marrow culture-initiating cells (LTC-IC). A maintenance or a slight (threefold) increase of week 6 CAFC/LTC-IC was found after one week of culture. To analyze the presence of BPC mediating in vivo engraftment, expanded CD34+ cells were transplanted into preirradiated NOD/SCID mice at various time points. Only CD34+ cells cultured for up to four days successfully engrafted murine bone marrow with human cells expressing myeloid or lymphoid progenitor phenotypes. In contrast, five- and seven-day expanded human BPC did not detectably engraft NOD/SCID mice. When FL, SCF and IL-3-supplemented cultures were performed for seven days on fibronectin-coated plastic, or when IL-3 was replaced by thrombopoietin, colony forming cells and LTC-IC reached levels similar to those of control cultures, yet no human cell engraftment was recorded in the mice. Also, culture in U-bottom microplates resulting in locally increased CD34+ cell density had no positive effect on engraftment. These results indicate that during ex vivo expansion of human CD34+ cells, CFC and LTC-IC numbers do not correlate with the potential to repopulate NOD/SCID mice. Our results suggest that ex vivo expanded BPC should be cultured for limited time periods only, in order to preserve bone-marrow-repopulating hematopoietic stem cells.     

Cobblestone area-forming cells in human cord blood are heterogeneous and differ from long-term culture-initiating cells

The long-term culture-initiating cell (LTC-IC) assay is a physiological approach to the quantitation of primitive human hematopoietic cells. The readout using identification of cobblestone area-forming cells (CAFC) has gained popularity over the LTC-IC readout where cells are subcultured in a colony-forming cell assay. However, comparing the two assays, cord blood (CB) mononuclear cell (MNC) samples were found to contain a higher frequency of CAFC than LTC-IC (126 +/- 83 versus 40 +/- 31 per 10(5) cells, p = 0.0001). Overall, 60% of week-5 cobblestones produced by CB MNC were not functional LTC-IC and were classified as "false." Separation of CB MNC using immunomagnetic columns showed that false cobblestones were CD34(-)/lineage(+). Purified CD34(+) cells, as expected, gave very similar readouts in the two assays, with 4,084 and 3,468/10(5) cells being CAFC and LTC-IC, respectively. CD34(-)/lineage(-) cells did not form cobblestones or become CD34(+) on stroma or in cytokine culture. Human CB MNC contain a population of mature lineage(+) cells, possibly mature T or B cells, which, although producing cobblestone areas (CA), are not functional LTC-IC. The CAFC readout by this method, therefore, is unreliable for estimation of primitive hematopoietic cells by limiting dilution analysis in whole human CB or MNC and also may not detect CD34(-) CA stem cells.     

The role of c-Mpl ligands in the expansion of cord blood hematopoietic progenitors

The major limitations to the widespread use of high-dose chemotherapy or radiotherapy followed by autologous or allogeneic transplantation are the scarcity of stem cell donors and the depletion of the autologous stem cell reservoir. Cord blood is a readily available source of stem cells, which, however, might be limited in number. For this reason, up to now, cord blood transplantation has been restricted to children. Therefore, a major goal for experimental and clinical hematology is the identification of mechanisms and conditions that support the expansion of transplantable hematopoietic stem cells. Two systems have been described to identify in vitro these progenitor cell populations in both mice and humans: A) long-term culture-initiating cells (LTC-IC), so named because of their ability to support the growth of hemopoietic colonies (colony-forming cell [CFC]) for five to six weeks when cocultured on stromal layers, and B) the generation of hematopoietic progenitors CFC from stroma-free liquid cultures for extended periods of time, which is another indirect evidence for the presence of primitive stem cells. The two systems detect largely overlapping but not identical cell populations of progenitor cells; thus, the identification of the growth factor requirements for the maintenance and amplification of both systems is relevant. The studies presented here demonstrate that CD34+ cord blood cells can be grown in stroma-free liquid cultures for extremely prolonged periods of time (up to six months). During such a period, hemopoietic precursors and committed progenitors belonging to all of the hematopoietic lineages are continuously and massively generated. Such a massive expansion is sustained by an increasingly larger expansion of primitive stem cells (CFU-BI and LTC-IC). The presence of both FL and thrombopoietin (TPO) was necessary and sufficient to support this phenomenon. The addition of KL +/- interleukin 6 (IL-6) does not appear to substantially modify the extent of LTC-IC expansion. FL and TPO appear to be two unique growth factors that preferentially support the self-renewal of primitive stem cells; the additional presence of KL and IL-6 seems to enhance the proliferative potential of at least a subpopulation of daughter stem cells which can undergo at least three differentiation pathways.     

Co-transplantation of human mesenchymal stem cells promotes human CD34+ cells engraftment in a dose-dependent fashion in NOD/SCID mice

Mesenchymal stem cells (MSCs) have recently been identified and characterized in humans. Moreover, MSC secrete cytokines that can support hematopoietic progenitor growth. In the present study, we evaluated whether the efficacy of hematopoietic stem cell transplantation is improved by their co-transplantation with MSC, and whether this is positively correlated with the dose of infused MSCs. Accordingly, irradiated NOD/SCID mice were transplanted with 1 x 10(5) human CD34+ cells in the presence or absence of culture expanded MSCs (1 x 10(6) or 5 x 10(6)). We evaluated human hematopoietic cell engraftment by flow cytometry and assessed MSC tissue distributions by fluorescence in situ hybridization. We found that CD45+ and CD34+ cell levels were significantly elevated in a dose-dependent manner in co-transplanted mice 4 weeks after transplantation. The engraftments of CD33+ and CD19+ cells also increased dose-dependently. However, the engraftment of CD3+ cells did not increase after co-transplantation with MSCs. Human Y chromosome+ cells were observed in multiple tissues and were more frequently observed in mice co-transplanted with 5 x 10(6) rather than 1 x 10(6) MSCs. These results suggest that MSCs are capable of enhancing hematopoietic cell engraftment and distribution in multiple organs in a dose-dependent fashion.     

Characterization, isolation, and differentiation of murine skin cells expressing hematopoietic stem cell markers

As the phenotype of adult dermal stem cells is still elusive, and the hematopoietic stem cell is one of the best-characterized stem cells in the body, we tested dermal cell suspensions, sections, and wholemounts in newborn and adult mice for hematopoietic stem cell marker expression. Phenotypic analysis revealed that a small population of CD45(+) cells and a large population of CD45(-) cells expressed CD34, CD117, and stem cell antigen-1 molecules. When cultivated in selected media supplemented with hematopoietic cytokines, total dermal cells, lineage(-), and/or highly enriched phenotypically defined cell subsets produced hematopoietic and nonhematopoietic colonies. When injected into lethally irradiated recipient mice, a small percentage of newborn dermal cells was able to migrate into hematopoietic tissues and the skin and survived through the 11-month monitoring period. Our ability to isolate a candidate autologous stem cell pool will make these cells ideal vehicles for genetic manipulation and gene therapy.     

In vitro proliferation potential of AC133 positive cells in peripheral blood

AC133 antigen is a novel marker for human hematopoietic stem/progenitor cells. In this study, we examined the expression and proliferation potential of AC133(+) cells obtained from steady-state peripheral blood (PB). The proportion of AC133(+) cells in the CD34(+) subpopulation of steady-state PB was significantly lower than that of cord blood (CB), although that of cytokine-mobilized PB was higher than that of CB. The proliferation potential of AC133(+)CD34(+) and AC133(-)CD34(+) cells was examined by colony-forming analysis and analysis of long-term culture-initiating cells (LTC-IC). Although the total number of colony-forming cells was essentially the same in the AC133(+)CD34(+) fraction as in the AC133(-)CD34(+) fraction, the proportion of LTC-IC was much higher in the AC133(+)CD34(+) fraction. Virtually no LTC-IC were detected in the AC133(-)CD34(+) fraction. In addition, the features of the colonies grown from these two fractions were quite different. Approximately 70% of the colonies derived from the AC133(+)CD34(+) fraction were granulocyte-macrophage colonies, whereas more than 90% of the colonies derived from the AC133(-)CD34(+) fraction were erythroid colonies. Furthermore, an ex vivo expansion study observed expansion of colony-forming cells only in the AC133(+)CD34(+) population, and not in the AC133(-)CD34(+) population. These findings suggest that to isolate primitive hematopoietic cells from steady-state PB, selection by AC133 expression is better than selection by CD34 expression.     

Brief report: efficient generation of hematopoietic precursors and progenitors from human pluripotent stem cell lines

By mimicking embryonic development of the hematopoietic system, we have developed an optimized in vitro differentiation protocol for the generation of precursors of hematopoietic lineages and primitive hematopoietic cells from human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSCs). Factors such as cytokines, extra cellular matrix components, and small molecules as well as the temporal association and concentration of these factors were tested on seven different human ESC and iPSC lines. We report the differentiation of up to 84% human CD45+ cells (average 41% ± 16%, from seven pluripotent lines) from the differentiation culture, including significant numbers of primitive CD45+/CD34+ and CD45+/CD34+/CD38- hematopoietic progenitors. Moreover, the numbers of hematopoietic progenitor cells generated, as measured by colony forming unit assays, were comparable to numbers obtained from fresh umbilical cord blood mononuclear cell isolates on a per CD45+ cell basis. Our approach demonstrates highly efficient generation of multipotent hematopoietic progenitors with among the highest efficiencies reported to date (CD45+/CD34+) using a single standardized differentiation protocol on several human ESC and iPSC lines. Our data add to the cumulating evidence for the existence of an in vitro derived precursor to the hematopoietic stem cell (HSC) with limited engrafting ability in transplanted mice but with multipotent hematopoietic potential. Because this protocol efficiently expands the preblood precursors and hematopoietic progenitors, it is ideal for testing novel factors for the generation and expansion of definitive HSCs with long-term repopulating ability.     

胎肝条件培养液体外诱导人骨髓间充质干细胞定向分化为造血细胞的研究

目的：探讨人骨髓间充质干细胞（hMSCs）体外造血分化潜能。方法： 选用孕12.5-14.5 d（12.5-14.5 dpc）的昆明小鼠，分别制备小鼠胎肝基质细胞条件培养液（FLSC-CM）及胚胎成纤维细胞饲养层（FD），将体外扩增的CD34-CD45-hMSCs分别接种于含FLSC-CM、FD和IL-6及SCF组合的培养体系中，培养7 d后，通过形态学、表型、粒-单/巨噬细胞系集落培养（CFU-GM）对分化细胞进行鉴定。结果： hMSCs与FLSC-CM共培养组产生的非贴壁细胞明显增多，形态类似于单核或小淋巴细胞，部分细胞可表达人造血细胞特异性表面分子（CD34和CD45），在含人粒-单集落刺激因子（GM-CSF）的甲基纤维素培养体系中能够形成CFU-GM，而FD和IL-6+SCF诱导组无上述作用。结论： FLSC-CM可诱导CD34-CD45-hMSCs分化为造血细胞，提示hMSCs具有体外造血分化潜能。     

A novel immunodeficient mouse model--RAG2 x common cytokine receptor gamma chain double mutants--requiring exogenous cytokine administration for human hematopoietic stem cell engraftment.

Gene transduction into immature human hematopoietic cells collected from umbilical cord blood, bone marrow, or mobilized peripheral blood cells could be useful for the treatment of genetic and acquired disorders of the hematopoietic system. Immunodeficient mouse models have been used frequently as recipients to assay the growth and differentiation of human hematopoietic stem/progenitor cells. Indeed, high levels of human cell engraftment were first reported in human/murine chimeras using NOD/SCID mice, which now are considered as the standard for these types of experiments. However, NOD/SCID mice have some clear disadvantages (including spontaneous tumor formation) that limit their general use. We have developed a new immunodeficient mouse model by combining recombinase activating gene-2 (RAG2) and common cytokine receptor gamma chain (gamma c) mutations. The RAG2-/-/gamma c- double mutant mice are completely alymphoid (T-, B-, NK-), show no spontaneous tumor formation, and exhibit normal hematopoietic parameters. Interestingly, human cord blood cell engraftment in RAG2-/-/gamma c- mice was greatly enhanced by the exogenous administration of human cytokines interleukin-(IL-3) granulocyte-macrophage colony-stimulating factor, (GM-CSF), and erythropoietin in contrast to the NOD/SCID model. This unique feature of the RAG2-/-/gamma c- mouse model should be particularly well suited for assessing the role of different cytokines in human lymphopoiesis and stem/progenitor cell function in vivo.     

High levels of transgene expression following transduction of long-term NOD/SCID-repopulating human cells with a modified lentiviral vector

Both oncoretroviral and lentiviral vectors have been shown to transduce CD34(+) human hematopoietic stem cells (HSC) capable of establishing human hematopoiesis in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice that support partially human hematopoiesis. We and others have reported that murine stem cell virus (MSCV)-based oncoretroviral vectors efficiently transduced HSC that had been cultured ex vivo for 4-7 days with cytokines, resulting in transgene expression in lymphoid and myeloid progenies of SCID-engrafting cells 4-8 weeks post-transplantation. Although lentiviral vectors have been demonstrated to transduce HSC under minimal ex vivo culture conditions, concerns exist regarding the level of transgene expression mediated by these vectors. We therefore evaluated a novel hybrid lentiviral vector (GIN-MU3), in which the U3 region of the HIV-1 long terminal repeat was replaced by the MSCV U3 region (MU3). Human cord blood CD34(+) cells were transduced with vesicular stomatitis virus G envelope protein-pseudotyped lentiviruses during a 48-hour culture period. After a total of 4 days in culture, transduced cells were transplanted into NOD/SCID mice to examine gene transfer and expression in engrafting human cells. Fifteen weeks post-transplantation, 37% +/- 12% of engrafted human cells expressed the green fluorescence protein (GFP) gene introduced by the lentiviral vector. High levels of GFP expression were observed in lymphoid, myeloid and erythroid progenies, and in engrafted human cells that retained the CD34(+) phenotype 15 weeks post-transplantation. This study provides evidence that lentiviral vectors transduced both short-term and long-term engrafting human cells, and mediated persistent transgene expression at high levels in multiple lineages of hematopoietic cells.     

Immune-deficient SCID and NOD/SCID mice models as functional assays for studying normal and malignant human hematopoiesis

 Many events and requirements of the developmental program of human hematopoietic stem cells have not yet been discovered. A major impediment has been the lack of an appropriate experimental system. At present the conditions for maintaining human stem cells in vitro are not fully known. As a result within a short period the small stem cell pool is lost due to differentiation, making it difficult to examine the correlation between these cells and their function in vivo. Most of our knowledge of hematopoietic stem cells is from animal models in which purified stem cell canididates are assayed based on their functional ability to rescue lethally conditioned recipients. The permanent correction of many genetic disorders of the hematopoietic system requires efficient methods for introducing genes into stem cells in vitro. However, progress has been hindered by the absence of preclinical models that assay the repopulating capacity of primitive human cells. In addition, the development of therapy for malignant diseases also requires assays to identify the target leukemic stem cells based on their ability to initiate the disease. The recent development of methods to transplant or implant both normal and leukemic cells into immune-deficient mice provides the foundation for human stem cell assays. These models assay the repopulating capacity of primitive human cells and provide an important approach to identify and characterize human stem cells, both normal and leukemic. This review focuses on the development of functional assays for normal and leukemic human stem cells and on the new insights that these models are beginning to provide on the organization of the human stem cell hierarchy. Received: 27 January 1997 / Accepted: 3 April 1997     

IFATS collection: Identification of hemangioblasts in the adult human adipose tissue

The stromal-vascular fraction (SVF) of human adipose tissue contains, among other cell types, mesenchymal stem cells and precursors of adipocyte and endothelial cells. Here we show that, in addition, the nonhematopoietic fraction of the SVF has hematopoietic activity, since all types of hematopoietic colony-forming units (CFUs) developed when cultured in methylcellulose-based medium. This hematopoietic activity was restricted to the CD45(-)CD105(+) cell subset, well correlated with KDR(+) cell content, and increased after culture with a combination of early-acting hematopoietic cytokines. Most of the CD45(-)KDR(+)CD105(+) cells were nonadherent and did not express CD31, and this subset included both CD34(-) and CD34(+) cells. Moreover, these nonadherent cells migrated in response to KDR gradient, and when they were cultured in the presence of both hematopoietic and endothelial growth factors, a wave of CFUs was followed by a wave of mixed colonies comprising adherent elongated and nonadherent round hematopoietic cells. These mixed hematopoietic-endothelial (Hem-End) colonies were able to generate secondary Hem-End colonies and exhibited both hematopoietic and endothelial activity, as demonstrated by in vitro functional assays. These findings demonstrate for the first time the existence of primitive mesodermal progenitors within the SVF of human adipose tissue that exhibit in vitro hematopoietic and hemangioblastic activities, susceptible to being used in cell therapy and basic cell research. Disclosure of potential conflicts of interest is found at the end of this article.     

Expansion of LTC-ICs and maintenance of p21 and BCL-2 expression in cord blood CD34(+)/CD38(-) early progenitors cultured over human MSCs as a feeder layer

Allogeneic transplantation with umbilical cord blood (UCB) is limited in adult recipients by a low CD34(+) cell dose. Clinical trials incorporating cytokine-based UCB in vitro expansion have not demonstrated significant shortening of hematologic recovery despite substantial increases in CD34(+) cell dose, suggesting loss of stem cell function. To sustain stem cell function during cytokine-based in vitro expansion, a feeder layer of human mesenchymal stem cells (MSCs) was incorporated in an attempt to mimic the stem cell niche in the marrow microenvironment. UCB expansion on MSCs resulted in a 7.7-fold increase in total LTC-IC output and a 3.8-fold increase of total early CD34(+) progenitors (CD38(-)/HLA-DR(-)). Importantly, early CD34(+)/CD38(-)/HLA-DR(-) progenitors from cultures expanded on MSCs demonstrated higher cytoplasmic expression of the cell-cycle inhibitor, p21(cip1/waf1), and the antiapoptotic protein, BCL-2, compared with UCB expanded in cytokines alone, suggesting improved maintenance of stem cell function in the presence of MSCs. Moreover, the presence of MSCs did not elicit UCB lymphocyte activation. Taken together, these results strongly suggest that the addition of MSCs as a feeder layer provides improved conditions for expansion of early UCB CD34(+)/CD38(-)/HLA-DR(-) hematopoietic progenitors and may serve to inhibit their differentiation and rates of apoptosis during short-term in vitro expansion.     

Characterization of chemokine receptors expressed in primitive blood cells during human hematopoietic ontogeny

Chemokines are capable of regulating a variety of fundamental processes of hematopoietic cells that include proliferation, differentiation, and migration. To evaluate potential chemokine signaling pathways important to the regulation of primitive human hematopoietic cells, we examined chemokine receptor expression of highly purified subpopulations of uncommitted human blood cells. CXCR1-, CXCR2-, CXCR4-, and CCR5-expressing cells were detected by flow cytometry among human blood subsets depleted of lineage-restricted cells (Lin(-)) derived from adult bone marrow, mobilized peripheral blood, cord blood (CB), and circulating fetal blood. Although these chemokine receptors could be detected on Lin(-) cells throughout human development, only CXCR4 could be detected in CD34(-)CD38(-)Lin(-) and CD34(+)CD38(-)Lin(-) subfractions enriched for stem cell function, suggesting that independent of ontogeny, CXCR4-mediated signals are critical to primitive hematopoiesis. Distinct to other stages of human hematopoietic development, primitive CB cells expressed higher levels of CXCR1, CXCR2, CCR5, and CXCR4 on both CD34(-)CD38(-)Lin(-) and CD34(+)CD38(-)Lin(-) subsets. Isolation of these fractions revealed expression of additional chemokine receptors CCR7, CCR8, and Bonzo (STRL133), whereas BOB (GPR15) could not be detected. Our study illustrates that rare uncommitted hematopoietic cells express chemokine receptors not previously associated with primitive human blood cells. Based on these results, we suggest that signaling pathways mediated by chemokine receptors identified here may play a fundamental role in hematopoietic stem cell regulation and provide alternative receptor targets for retroviral pseudotyping for genetic modification of repopulating cells.     

人骨髓间质干细胞向造血细胞分化潜能的实验研究

目的:在体研究人骨髓间质干细胞(hBMMSCs)向造血细胞分化的潜能。方法:将hBMMSCs经尾静脉注射给环磷酰胺处理的严重联合免疫缺陷(SCID)小鼠,利用流式激活细胞分析系统(FACS)检测hBMMSCs输注后存活35d的SCID小鼠外周血、骨髓和脾脏中人源性造血细胞的表型和水平。结果:hBMMSCs输注组外周血(PB)、骨髓(BM)和脾脏(spleen)中可检测到人CD45⁺/H-2D^d-、CD34⁺/H-2D^d-细胞,而对照组PB、BM和脾脏均未检测到上述表型的人造血细胞。结论:hBMMSCs具有向造血细胞分化的潜能。     

Mesenchymal stem cells secreting angiopoietin-like-5 support efficient expansion of human hematopoietic stem cells without compromising their repopulating potential

Clinical and preclinical applications of human hematopoietic stem cells (HSCs) are often limited by scarcity of cells. Expanding human HSCs to increase their numbers while maintaining their stem cell properties has therefore become an important area of research. Here, we report a robust HSC coculture system wherein cord blood CD34(+) CD133(+) cells were cocultured with mesenchymal stem cells engineered to express angiopoietin-like-5 in a defined medium. After 11 days of culture, SCID repopulating cells were expanded ~60-fold by limiting dilution assay in NOD-scid Il2rg(-/-) (NSG) mice. The cultured CD34(+) CD133(+) cells had similar engraftment potential to uncultured CD34(+) CD133(+) cells in competitive repopulation assays and were capable of efficient secondary reconstitution. Further, the expanded cells supported a robust multilineage reconstitution of human blood cells in NSG recipient mice, including a more efficient T-cell reconstitution. These results demonstrate that the expanded CD34(+) CD133(+) cells maintain both short-term and long-term HSC activities. To our knowledge, this ~60-fold expansion of SCID repopulating cells is the best expansion of human HSCs reported to date. Further development of this coculture method for expanding human HSCs for clinical and preclinical applications is therefore warranted.     

人胎盘绒毛膜来源间充质干细胞的生物学特性

背景：骨髓是间充质干细胞的主要来源,但骨髓中间充质干细胞数量和质量会随着年龄的增长而逐渐降低。因此,寻找一种新的干细胞来源具有重要意义。 目的：分离研究了一种新的间充质干细胞来源——人绒毛膜来源间充质干细胞,并研究其生物学特性。 方法：将胎盘冲洗干净后,分离出脐带和羊膜组织,将剩余的胎盘组织进行原代和传代培养。通过短串联重复序列分析检测细胞是否来源于绒毛膜,用MTT法检测细胞生长方式,流式细胞仪分析细胞表型及干细胞标记,使用不同的诱导分化培养基检测其多向分化的能力。将该细胞与植物血凝素刺激的外周血单个核细胞共培养,用酶联免疫吸附试验检测上清γ-干扰素的水平。 结果与结论：短串联重复序列分析证明所得到的细胞来源于绒毛膜,这种绒毛膜来源的细胞生长呈典型的成纤维细胞形态。细胞表达常见的间充质干细胞表面标记CD90、CD73、CD105、CD44,不表达CD45,CD11b和CD34。同时,细胞也表达Nestin和Sox-2。在不同的条件培养基培养状态下,细胞可向成骨、成脂方向分化。这些结果证明所得到的细胞为人绒毛膜间充质干细胞。这些绒毛膜间充质干细胞能抑制植物血凝素刺激的人外周血单个核细胞分泌γ-干扰素。可见绒毛膜来源的间充质干细胞具有和传统的骨髓来源间充质干细胞具有相似的生物学特性。     

Dengue virus infection and immune response in humanized RAG2(-/-)gamma(c)(-/-) (RAG-hu) mice

Dengue viral (DENV) pathogenesis and vaccine studies are hampered by the lack of an ideal animal model mimicking human disease and eliciting an adaptive human immune response. Although currently available animal models have been very useful in dissecting some key aspects of disease pathogenesis, a major limitation with these is the lack of a human immune response. In this study, we sought to overcome this difficulty by utilizing a novel mouse model that permits multi-lineage human hematopoiesis and immune response following transplantation with human hematopoietic stem cells. To generate immunocompetent humanized mice, neonatal RAG2(-/-)gamma(c)(-/-) mice were xenografted with human CD34+ hematopoietic stem cells, resulting in de novo development of major functional cells of the human adaptive immune system. To evaluate susceptibility to dengue viral infection, humanized mice were challenged with DEN-2 serotype. Viremia lasting up to 3 weeks was detected in infected mice with viral titers reaching up to 10(6.3) RNA copies/ml. Fever characteristic of dengue was also noted in infected mice. Presence of human anti-dengue antibodies was evaluated using an antibody capture ELISA. Anti-dengue IgM was first detected by 2 weeks post-infection followed by IgG at 6 weeks. Sera from some of the infected mice were also found to be capable of dengue virus neutralization. Infected mouse sera showed reactivity with the viral envelope and capsid proteins in immunoprecipitation assay. These results demonstrate for the first time that humanized mice are capable of dengue viral primary human immune responses thus paving the way for new dengue immunopathogenesis and vaccine studies.     

Endotoxin hyperresponsiveness upon CD4+ T cell reconstitution in lymphopenic mice: control by natural regulatory T cells

Natural CD4(+)CD25(+) regulatory T cells (nTreg) have been shown to control graft-versus-host disease after hematopoietic stem cell transplantation (HSCT). Herein, we considered the possibility that the beneficial action of nTreg upon immune reconstitution in lymphopenic hosts involves dampening of the inflammatory response induced by bacterial products. We first observed that transfer of syngeneic CD4(+)CD25(-) T cells in RAG-deficient mice dramatically enhanced release of inflammatory cytokines and associated pathology upon endotoxin injection. Interferon (IFN)-gamma produced by T cells undergoing homeostatic proliferation was shown to be involved in the endotoxin hyperresponsiveness induced by CD4(+) T cell reconstitution. Co-transfer of CD4(+)CD25(+) nTreg with CD4(+)CD25(-) T cells inhibited the expansion of IFN-gamma-producing T cells and reduced endotoxin responses in RAG(-/-) mice. We conclude that (1) CD4(+) T cell reconstitution sensitizes lymphopenic hosts to endotoxin-induced pathology and (2) nTreg prevent this process by limiting the emergence of IFN-gamma-producing cells.