The HOXB4 homeoprotein promotes the ex vivo enrichment of functional human embryonic stem cell-derived NK cells

Human embryonic stem cells (hESCs) can be induced to differentiate into blood cells using either co-culture with stromal cells or following human embryoid bodies (hEBs) formation. It is now well established that the HOXB4 homeoprotein promotes the expansion of human adult hematopoietic stem cells (HSCs) but also myeloid and lymphoid progenitors. However, the role of HOXB4 in the development of hematopoietic cells from hESCs and particularly in the generation of hESC-derived NK-progenitor cells remains elusive. Based on the ability of HOXB4 to passively enter hematopoietic cells in a system that comprises a co-culture with the MS-5/SP-HOXB4 stromal cells, we provide evidence that HOXB4 delivery promotes the enrichment of hEB-derived precursors that could differentiate into fully mature and functional NK. These hEB-derived NK cells enriched by HOXB4 were characterized according to their CMH class I receptor expression, their cytotoxic arsenal, their expression of IFNγ and CD107a after stimulation and their lytic activity. Furthermore our study provides new insights into the gene expression profile of hEB-derived cells exposed to HOXB4 and shows the emergence of CD34(+)CD45RA(+) precursors from hEBs indicating the lymphoid specification of hESC-derived hematopoietic precursors. Altogether, our results outline the effects of HOXB4 in combination with stromal cells in the development of NK cells from hESCs and suggest the potential use of HOXB4 protein for NK-cell enrichment from pluripotent stem cells.     

Human pluripotent stem cells produce natural killer cells that mediate anti-HIV-1 activity by utilizing diverse cellular mechanisms

Cell-based therapies against HIV/AIDS have been gaining increased interest. Natural killer (NK) cells are a key component of the innate immune system with the ability to kill diverse tumor cells and virus-infected cells. While NK cells have been shown to play an important role in the control of HIV-1 replication, their functional activities are often compromised in HIV-1-infected individuals. We have previously demonstrated the derivation of NK cells from human embryonic stem cells (hESCs) with the ability to potently kill multiple types of tumor cells both in vitro and in vivo. We now demonstrate the derivation of functional NK cells from human induced pluripotent stem cells (iPSCs). More importantly, both hESC- and iPSC-derived NK cells are able to inhibit HIV-1 NL4-3 infection of CEM-GFP cells. Additional studies using HIV-1-infected human primary CD4(+) T cells illustrated that hESC- and iPSC-derived NK cells suppress HIV-1 infection by at least three distinct cellular mechanisms: killing of infected targets through direct lysis, antibody-dependent cellular cytotoxicity, and production of chemokines and cytokines. Our results establish the potential to utilize hESC- and iPSC-derived NK cells to better understand anti-HIV-1 immunity and provide a novel cellular immunotherapeutic approach to treat HIV/AIDS.     

Specific marking of hESCs-derived hematopoietic lineage by WAS-promoter driven lentiviral vectors

Genetic manipulation of human embryonic stem cells (hESCs) is instrumental for tracing lineage commitment and to studying human development. Here we used hematopoietic-specific Wiskott-Aldrich syndrome gene (WAS)-promoter driven lentiviral vectors (LVs) to achieve highly specific gene expression in hESCs-derived hematopoietic cells. We first demonstrated that endogenous WAS gene was not expressed in undifferentiated hESCs but was evident in hemogenic progenitors (CD45(-)CD31(+)CD34(+)) and hematopoietic cells (CD45(+)). Accordingly, WAS-promoter driven LVs were unable to express the eGFP transgene in undifferentiated hESCs. eGFP(+) cells only appeared after embryoid body (EB) hematopoietic differentiation. The phenotypic analysis of the eGFP(+) cells showed marking of different subpopulations at different days of differentiation. At days 10-15, AWE LVs tag hemogenic and hematopoietic progenitors cells (CD45(-)CD31(+)CD34(dim) and CD45(+)CD31(+)CD34(dim)) emerging from hESCs and at day 22 its expression became restricted to mature hematopoietic cells (CD45(+)CD33(+)). Surprisingly, at day 10 of differentiation, the AWE vector also marked CD45(-)CD31(low/-)CD34(-) cells, a population that disappeared at later stages of differentiation. We showed that the eGFP(+)CD45(-)CD31(+) population generate 5 times more CD45(+) cells than the eGFP(-)CD45(-)CD31(+) indicating that the AWE vector was identifying a subpopulation inside the CD45(-)CD31(+) cells with higher hemogenic capacity. We also showed generation of CD45(+) cells from the eGFP(+)CD45(-)CD31(low/-)CD34(-) population but not from the eGFP(-)CD45(-)CD31(low/-)CD34(-) cells. This is, to our knowledge, the first report of a gene transfer vector which specifically labels hemogenic progenitors and hematopoietic cells emerging from hESCs. We propose the use of WAS-promoter driven LVs as a novel tool to studying human hematopoietic development.     

Characterization of developmental pathway of natural killer cells from embryonic stem cells in vitro

In vitro differentiation of embryonic stem (ES) cells is often used to study hematopoiesis. However, the differentiation pathway of lymphocytes, in particular natural killer (NK) cells, from ES cells is still unclear. Here, we used a multi-step in vitro ES cell differentiation system to study lymphocyte development from ES cells, and to characterize NK developmental intermediates. We generated embryoid bodies (EBs) from ES cells, isolated CD34(+) EB cells and cultured them on OP9 stroma with a cocktail of cytokines to generate cells we termed ES-derived hematopoietic progenitors (ES-HPs). EB cell subsets, as well as ES-HPs derived from EBs, were tested for NK, T, B and myeloid lineage potentials using lineage specific cultures. ES-HPs derived from CD34(+) EBs differentiated into NK cells when cultured on OP9 stroma with IL-2 and IL-15, and into T cells on Delta-like 1-transduced OP9 (OP9-DL1) with IL-7 and Flt3-L. Among CD34(+) EB cells, NK and T cell potentials were detected in a CD45(-) subset, whereas CD45(+) EB cells had myeloid but not lymphoid potentials. Limiting dilution analysis of ES-HPs generated from CD34(+)CD45(-) EB cells showed that CD45(+)Mac-1(-)Ter119(-) ES-HPs are highly enriched for NK progenitors, but they also have T, B and myeloid potentials. We concluded that CD45(-)CD34(+) EB cells have lymphoid potential, and they differentiate into more mature CD45(+)Lin(-) hematopoietic progenitors that have lymphoid and myeloid potential. NK progenitors among ES-HPs are CD122(-) and they rapidly acquire CD122 as they differentiate along the NK lineage.     

Fibroblasts from human spleen regulate NK cell differentiation from blood CD34(+) progenitors via cell surface IL-15

Besides a structural role in tissue architecture, fibroblasts have been shown to regulate the proliferation and differentiation of other neighboring specialized cell types, but differently according to the anatomic site and pathologic status of their tissue of origin. In this study we report a novel regulatory function of human spleen-derived fibroblasts in the development of NK cells from adult resting blood progenitors. When CD34(+) cells were cocultured with spleen-derived fibroblasts in monolayers, nonadherent CD56(+)CD3(-) NK cells were predominantly produced after 2-3 wk of culture in the absence of exogenous cytokines. Most NK cells expressed class I-recognizing CD94 and NK p46, p44, and p30 receptors as well as perforin and granzyme lytic granules. Moreover, these cells demonstrated spontaneous killing activity. Cell surface immunophenotyping of spleen-derived fibroblasts revealed a low and consistent expression of IL-15, Flt3 ligand, and c-kit ligand. Additionally, low picogram amounts of the three cytokines were produced extracellularly. Neutralizing Abs to IL-15, but not the other two ligands, blocked NK cell development. Additionally, suppressing direct contacts of CD34(+) progenitors and fibroblasts by microporous membrane abrogated NK cell production. We conclude that stromal fibroblasts within the human spleen are involved via constitutive cell surface expression of bioactive IL-15 in the development of functional activated NK cells under physiologic conditions.     

TGFβ inhibition enhances the generation of hematopoietic progenitors from human ES cell-derived hemogenic endothelial cells using a stepwise strategy

Embryonic hematopoiesis is a complex process. Elucidating the mechanism regulating hematopoietic differentiation from pluripotent stem cells would allow us to establish a strategy to efficiently generate hematopoietic cells. However, the mechanism governing the generation of hematopoietic progenitors from human embryonic stem cells (hESCs) remains unknown. Here, on the basis of the emergence of CD43(+) hematopoietic cells from hemogenic endothelial (HE) cells, we demonstrated that VEGF was essential and sufficient, and that bFGF was synergistic with VEGF to specify the HE cells and the subsequent transition into CD43(+) hematopoietic cells. Significantly, we identified TGFβ as a novel signal to regulate hematopoietic development, as the TGFβ inhibitor SB 431542 significantly promoted the transition from HE cells into CD43(+) hematopoietic progenitor cells (HPCs) during hESC differentiation. By defining these critical signaling factors during hematopoietic differentiation, we can efficiently generate HPCs from hESCs. Our strategy could offer an in vitro model to study early human hematopoietic development.     

Improved development of human embryonic stem cell-derived embryoid bodies by stirred vessel cultivation

Human embryonic stem cells (hESCs) represent an important resource for novel cell-based regenerative medical therapies. hESCs are known to differentiate into mature cells of defined lineages through the formation of embryoid bodies (EBs) which are amenable to suspension culture for several weeks. However, EBs derived from hESCs in standard static cultures are typically non-homogeneous, leading to inefficient cellular development. Here, we systematically compare the formation, growth, and differentiation capabilities of hESC-derived EBs in stirred and static suspension cultures. A 15-fold expansion in total number of EB-derived cells cultured for 21 days in a stirred flask was observed, compared to a fourfold expansion in static (non-stirred) cultures. Additionally, stirred vessel mediated cultures have a more homogeneous EB morphology and size. Importantly, the EBs cultivated in spinner flasks retained comparable ability to produce hematopoietic progenitor cells as those grown in static culture. These results demonstrate the decoupling between EB cultivation method and EB-derived cells' ability to form hematopoietic progenitors, and will allow for improved production of scalable quantities of hematopoietic cells or other differentiated cell lineages from hESCs in a controlled environment.     

Human NK cells: From development to effector functions

NK cells are the major lymphocyte subset of the innate immune system that mediates antiviral and anti-tumor responses. It is well established that they develop mechanisms to distinguish self from non-self during the process of NK cell education. Unlike T and B cells, natural killer cells lack clonotypic receptors and are activated after recognizing their target via germline-encoded receptors through natural cytotoxicity, cytokine stimulation, and Ab-dependent cellular cytotoxicity. Subsequently, they utilize cytotoxic granules, death receptor ligands, and cytokines to perform their effector functions. In this review, we provide a general overview of human NK cells, as opposed to murine NK cells, discussing their ontogeny, maturation, receptor diversity, types of responses, and effector functions. Furthermore, we also describe recent advances in human NK cell biology, including tissue-resident NK cell populations, NK cell memory, and novel approaches used to target NK cells in cancer immunotherapy.     

Selective expansion and partial activation of human NK cells and NK receptor-positive T cells by IL-2 and IL-15.

IL-2 and IL-15 are lymphocyte growth factors produced by different cell types with overlapping functions in immune responses. Both cytokines costimulate lymphocyte proliferation and activation, while IL-15 additionally promotes the development and survival of NK cells, NKT cells, and intraepithelial lymphocytes. We have investigated the effects of IL-2 and IL-15 on proliferation, cytotoxicity, and cytokine secretion by human PBMC subpopulations in vitro. Both cytokines selectively induced the proliferation of NK cells and CD56(+) T cells, but not CD56(-) lymphocytes. All NK and CD56(+) T cell subpopulations tested (CD4(+), CD8(+), CD4(-)CD8(-), alphabetaTCR(+), gammadeltaTCR(+), CD16(+), CD161(+), CD158a(+), CD158b(+), KIR3DL1(+), and CD94(+)) expanded in response to both cytokines, whereas all CD56(-) cell subpopulations did not. Therefore, previously reported IL-15-induced gammadelta and CD8(+) T cell expansions reflect proliferations of NK and CD56(+) T cells that most frequently express these phenotypes. IL-15 also expanded CD8alpha(+)beta(-) and Valpha24Vbeta11 TCR(+) T cells. Both cytokines stimulated cytotoxicity by NK and CD56(+) T cells against K562 targets, but not the production of IFN-gamma, TNF-alpha, IL-2, or IL-4. However, they augmented cytokine production in response to phorbol ester stimulation or CD3 cross-linking by inducing the proliferation of NK cells and CD56(+) T cells that produce these cytokines at greater frequencies than other T cells. These results indicate that IL-2 and IL-15 act at different stages of the immune response by expanding and partially activating NK receptor-positive lymphocytes, but, on their own, do not influence the Th1/Th2 balance of adaptive immune responses.     

Human thymic epithelial cells inhibit IL-15- and IL-2-driven differentiation of NK cells from the early human thymic progenitors

T/NK progenitors are present in the thymus; however, the thymus predominantly promotes T cell development. In this study, we demonstrated that human thymic epithelial cells (TEC) inhibit NK cell development. Most ex vivo human thymocytes express CD1a, indicating that thymic progenitors are predominantly committed to the T cell lineage. In contrast, the CD1a(-)CD3(-)CD56(+) NK population comprises only 0.2% (n = 7) of thymocytes. However, we observed increases in the percentage (20- to 25-fold) and absolute number (13- to 71-fold) of NK cells when thymocytes were cultured with mixtures of either IL-2, IL-7, and stem cell factor or IL-15, IL-7, and stem cell factor. TEC, when present in the cultures, inhibited the increases in the percentage (3- to 10-fold) and absolute number (3- to 25-fold) of NK cells. Furthermore, we show that TEC-derived soluble factors inhibit generation of NK-CFU and inhibit IL15- or IL2-driven NK cell differentiation from thymic CD34(+) triple-negative thymocytes. The inhibitory activity was found to be associated with a 8,000- to 30,000 Da fraction. Thus, our data demonstrate that TEC inhibit NK cell development from T/NK CD34(+) triple negative progenitors via soluble factor(s), suggesting that the human thymic microenvironment not only actively promotes T cell maturation but also controls the development of non-T lineage cells such as the NK lineage.     

Adoptive transfer of gene-modified primary NK cells can specifically inhibit tumor progression in vivo

NK cells hold great potential for improving the immunotherapy of cancer. Nevertheless, tumor cells can effectively escape NK cell-mediated apoptosis through interaction of MHC molecules with NK cell inhibitory receptors. Thus, to harness NK cell effector function against tumors, we used Amaxa gene transfer technology to gene-modify primary mouse NK cells with a chimeric single-chain variable fragment (scFv) receptor specific for the human erbB2 tumor-associated Ag. The chimeric receptor was composed of the extracellular scFv anti-erbB2 Ab linked to the transmembrane and cytoplasmic CD28 and TCR-zeta signaling domains (scFv-CD28-zeta). In this study we demonstrated that mouse NK cells gene-modified with this chimera could specifically mediate enhanced killing of an erbB2(+) MHC class I(+) lymphoma in a perforin-dependent manner. Expression of the chimera did not interfere with NK cell-mediated cytotoxicity mediated by endogenous NK receptors. Furthermore, adoptive transfer of gene-modified NK cells significantly enhanced the survival of RAG mice bearing established i.p. RMA-erbB2(+) lymphoma. In summary, these data suggest that use of genetically modified NK cells could broaden the scope of cancer immunotherapy for patients.     

Broad distribution of colony-forming cells with erythroid, myeloid, dendritic cell, and NK cell potential among CD34(++) fetal liver cells.

The generation of erythroid, myeloid, and lymphoid cells from human fetal liver progenitors was studied in colony-forming cell (CFC) assays. CD38(-) and CD38(+) progenitors that expressed high levels of CD34 were grown in serum-deprived medium supplemented with kit ligand, flk2/flt3 ligand, GM-CSF, c-mpl ligand, erythropoietin, and IL-15. The resulting colonies were individually analyzed by flow cytometry. CD56(+) NK cells were detected in 21.9 and 9.9% of colonies grown from CD38(-) and CD38(+) progenitors, respectively. NK cells were detected in mostly large CD14(+)/CD15(+) myeloid colonies that also, in some cases, contained red cells. NK cells were rarely detected in erythroid colonies, suggesting an early split between the erythroid and the NK cell lineages. CD1a(+) dendritic cells were also present in three-quarters of the colonies grown from CD38(-) and CD38(+) progenitors. Multilineage colonies containing erythrocytes, myeloid cells, and NK cells were present in 13.7 and 2.7% of colonies grown from CD38(-) and CD38(+) progenitors, respectively. High proliferative-potential CFCs that generated multilineage colonies were also detected among both populations of progenitors. The total number of high proliferative-potential CFCs with erythroid, myeloid, and NK cell potential was estimated to be 2-fold higher in the CD38(+) fraction compared with the CD38(-) fraction because of the higher frequency of CD38(+) cells among CD34(++) cells. The broad distribution of multipotent CFCs among CD38(-) and CD38(+) progenitors suggests that the segregation of the erythroid, myeloid, and lymphoid lineages may not always be an early event in hemopoiesis. Alternatively, some stem cells may be present among CD38(+) cells.     

Prethymic T-cell development defined by the expression of paired immunoglobulin-like receptors

T cells are produced in the thymus from progenitors of extrathymic origin. As no specific markers are available, the developmental pathway of progenitors preceding thymic colonization remains unclear. Here we show that progenitors in murine fetal liver and blood, which are capable of giving rise to T cells, NK cells and dendritic cells, but not B cells, can be isolated by their surface expression of paired immunoglobulin-like receptors (PIR). PIR expression is maintained until the earliest intrathymic stage, then downregulated before the onset of CD25 expression. Unlike intrathymic progenitors, generation of prethymic PIR(+) progenitors does not require Hes1-mediated Notch signaling. These findings disclose a prethymic stage of T-cell development programmed for immigration of the thymus, which is genetically separable from intrathymic stages.     

Genetic approach to track neural cell fate decisions using human embryonic stem cells

With their capability to undergo unlimited self-renewal and to differentiate into all cell types in the body, human embryonic stem cells (hESCs) hold great promise in human cell therapy. However, there are limited tools for easily identifying and isolating live hESC-derived cells. To track hESC-derived neural progenitor cells (NPCs), we applied homologous recombination to knock-in the mCherry gene into the Nestin locus of hESCs. This facilitated the genetic labeling of Nestin positive neural progenitor cells with mCherry. Our reporter system enables the visualization of neural induction from hESCs both in vitro (embryoid bodies) and in vivo (teratomas). This system also permits the identification of different neural subpopulations based on the intensity of our fluorescent reporter. In this context, a high level of mCherry expression showed enrichment for neural progenitors, while lower mCherry corresponded with more committed neural states. Combination of mCherry high expression with cell surface antigen staining enabled further enrichment of hESC-derived NPCs. These mCherry⁺NPCs could be expanded in culture and their differentiation resulted in a down-regulation of mCherry consistent with the loss of Nestin expression. Therefore, we have developed a fluorescent reporter system that can be used to trace neural differentiation events of hESCs.     

CCR7 ligands, SLC/6Ckine/Exodus2/TCA4 and CKbeta-11/MIP-3beta/ELC, are chemoattractants for CD56(+)CD16(-) NK cells and late stage lymphoid progenitors

Two human CC chemokines, SLC/6Ckine/Exodus2/TCA4 and CKbeta-11/MIP-3beta/ELC, are previously reported as efficacious chemoattractants for T- and B-cells and dendritic cells. SLC and CKbeta-11 share only 32% amino acid identity, but are ligands for the same chemokine receptor, CCR7. In this study, we examined chemotactic activity of SLC and CKbeta-11 for NK cells and lymphoid progenitors in bone marrow and thymus. It was found that these two CCR7 ligands are chemoattractants for neonatal cord blood and adult peripheral blood NK cells and cell lines. SLC and CKbeta-11 preferentially attract the CD56(+)CD16(-) NK cell subset over CD56(+)CD16(+) NK cells. SLC and CKbeta-11 also demonstrate selective chemotactic activity on late stage CD34(-)CD19(+)IgM- B-cell progenitors and CD4(+) and CD8(+) single-positive thymocytes, but not early stage progenitors. It was noted that SLC is an efficient desensitizer of CKbeta-11-dependent NK cell chemotaxis, while CKbeta-11 is a weak desensitizer of SLC-dependent chemotaxis. Taken together, these results suggest that SLC and CKbeta-11 have the potential to control trafficking of NK cell subsets and late stage lymphoid progenitors in bone marrow and thymus.     

T/NK bipotent progenitors in the thymus retain the potential to generate dendritic cells

We have previously shown that the earliest thymic progenitors retain the potential to generate T and NK cells and that they lose the bipotentiality to give rise to unipotent T and NK progenitors during the progression of intrathymic developmental stages. The present study examines the ability of these thymic progenitors for generation of dendritic cells (DC) with a new clonal assay that is capable of determining the developmental potential for DC in addition to T cells and NK cells. We found that the large majority of the T/NK bipotential progenitors in the earliest population of fetal thymus was able to generate DC. Although the DC potential is lost with the progression of the differentiation stage, some of the T/NK bipotential progenitors still retain their DC potential even at the CD44(+)CD25(+) stage.