In vitro and in vivo differentiation into B cells, T cells, and myeloid cells of primitive yolk sac hematopoietic precursor cells expanded >100-fold by coculture with a clonal yolk sac endothelial cell line

The yolk sac, first site of hematopoiesis during mammalian development, contains not only hematopoietic stem cells but also the earliest precursors of endothelial cells. We have previously shown that a nonadherent yolk sac cell population (WGA+, density <1.077, AA4.1+) can give rise to B cells, T cells, and myeloid cells both in vitro and in vivo. We now report on the ability of a yolk sac-derived cloned endothelial cell line (C166) to provide a suitable microenvironment for expansion of these early precursor cells. Single day 10 embryonic mouse yolk sac hematopoietic stem cells were expanded >100 fold within 8 days by coculture with irradiated C166 cells. Colony-forming ability was retained for at least three passages in vitro, with retention of the ability to differentiate into T-cell, B-cell, and myeloid lineages. Stem cell properties were maintained by a significant fraction of nonadherent cells in the third passage, although these stem cells expressed a somewhat more mature cell surface phenotype than the initial yolk sac stem cells. When reintroduced into adult allogeneic immunocompromised (scid) hosts, they were able to give rise to all of the leukocyte lineages, including T cells, B cells, and myeloid cells. We conclude that yolk sac endothelial cells can support the stable proliferation of multipotential hematopoietic stem cells, thus generating adequate numbers of cells for study of the mechanisms involved in their subsequent development and differentiation, for in vivo hematopoietic restitution, and for potential use as a vehicle for gene transfer.

Production of zebrafish germ-line chimeras from embryo cell cultures

Although the zebrafish possesses many characteristics that make it a valuable model for genetic studies of vertebrate development, one deficiency of this model system is the absence of methods for cell-mediated gene transfer and targeted gene inactivation. In mice, embryonic stem cell cultures are routinely used for gene transfer and provide the advantage of in vitro selection for rare events such as homologous recombination and targeted mutation. Transgenic animals possessing a mutated copy of the targeted gene are generated when the selected cells contribute to the germ line of a chimeric embryo. Although zebrafish embryo cell cultures that exhibit characteristics of embryonic stem cells have been described, successful contribution of the cells to the germ-cell lineage of a host embryo has not been reported. In this study, we demonstrate that short-term zebrafish embryo cell cultures maintained in the presence of cells from a rainbow trout spleen cell line (RTS34st) are able to produce germ-line chimeras when introduced into a host embryo. Messenger RNA encoding the primordial germ-cell marker, vasa, was present for more than 30 days in embryo cells cocultured with RTS34st cells or their conditioned medium and disappeared by 5 days in the absence of the spleen cells. The RTS34st cells also inhibited melanocyte and neuronal cell differentiation in the embryo cell cultures. These results suggest that the RTS34st splenic–stromal cell line will be a valuable tool in the development of a cell-based gene transfer approach to targeted gene inactivation in zebrafish.

Somatic mosaicism in Fanconi anemia: Evidence of genotypic reversion in lymphohematopoietic stem cells

Somatic mosaicism has been observed previously in the lymphocyte population of patients with Fanconi anemia (FA). To identify the cellular origin of the genotypic reversion, we examined each lymphohematopoietic and stromal cell lineage in an FA patient with a 2815–2816ins19 mutation in FANCA and known lymphocyte somatic mosaicism. DNA extracted from individually plucked peripheral blood T cell colonies and marrow colony-forming unit granulocyte–macrophage and burst-forming unit erythroid cells revealed absence of the maternal FANCA exon 29 mutation in 74.0%, 80.3%, and 86.2% of colonies, respectively. These data, together with the absence of the FANCA exon 29 mutation in Epstein–Barr virus-transformed B cells and its presence in fibroblasts, indicate that genotypic reversion, most likely because of back mutation, originated in a lymphohematopoietic stem cell and not solely in a lymphocyte population. Contrary to a predicted increase in marrow cellularity resulting from reversion in a hematopoietic stem cell, pancytopenia was progressive. Additional evaluations revealed a partial deletion of 11q in 3 of 20 bone marrow metaphase cells. By using interphase fluorescence in situ hybridization with an MLL gene probe mapped to band 11q23 to identify colony-forming unit granulocyte–macrophage and burst-forming unit erythroid cells with the 11q deletion, the abnormal clone was exclusive to colonies with the FANCA exon 29 mutation. Thus, we demonstrate the spontaneous genotypic reversion in a lymphohematopoietic stem cell. The subsequent development of a clonal cytogenetic abnormality in nonrevertant cells suggests that ex vivo correction of hematopoietic stem cells by gene transfer may not be sufficient for providing life-long stable hematopoiesis in patients with FA.

Role of MEKK2-MEK5 in the regulation of TNF-α gene expression and MEKK2-MKK7 in the activation of c-Jun N-terminal kinase in mast cells

Cross-linking of the high-affinity IgE receptor (FcɛRI) on mast cells with IgE and multivalent antigen triggers mitogen-activated protein (MAP) kinase activation and cytokine gene expression. We report here that MAP kinase kinase 4 (MKK4) gene disruption does not affect either MAP kinase activation or cytokine gene expression in response to cross-linking of FcɛRI in embryonic stem cell-derived mast cells. MKK7 is activated in response to cross-linking of FcɛRI, and this activation is inhibited by MAP/ERK kinase (MEK) kinase 2 (MEKK2) gene disruption. In addition, expression of kinase-inactive MKK7 in the murine mast cell line MC/9 inhibits c-Jun NH2-terminal kinase (JNK) activation in response to cross-linking of FcɛRI, whereas expression of kinase-inactive MKK4 does not affect JNK activation by this stimulus. However, FcɛRI-induced activation of the tumor necrosis factor-α (TNF-α) gene promoter is not affected by expression of kinase-inactive MKK7. We describe an alternative pathway by which MEKK2 activates MEK5 and big MAP kinase1/extracellular signal-regulated kinase 5 in addition to MKK7 and JNK, and interruption of this pathway inhibits TNF-α promoter activation. These findings suggest that JNK activation by antigen cross-linking is dependent on the MEKK2-MKK7 pathway, and cytokine production in mast cells is regulated in part by the signaling complex MEKK2-MEK5-ERK5.

Thrombopoietic potential and serial repopulating ability of murine hematopoietic stem cells constitutively expressing interleukin 11.

Based on transplantation studies with bone marrow cultured under various conditions, a role of interleukin 11 (IL-11) in the self-renewal and/or the differentiation commitment of hematopoietic stem cells has been indicated. To better evaluate the in vivo effects of IL-11 on stem/progenitor cell biology, lethally irradiated mice were serially transplanted with bone marrow cells transduced with a defective retrovirus, termed MSCV-mIL-11, carrying the murine IL-11 (mIL-11) cDNA and the bacterial neomycin phosphotransferase (neo) gene. High serum levels (i.e., > 1 ng/ml) of mIL-11 in all (20/20) primary and 86% (12/14) of secondary long-term reconstituted mice, as well as 86% (12/14) of tertiary recipients examined at 6 weeks posttransplant, demonstrated persistence of vector expression subsequent to transduction of bone marrow precursors functionally definable as totipotent hematopoietic stem cells. In agreement with results obtained with human IL-11 in other myeloablation models, ectopic mIL-11 expression accelerated recovery of platelets, neutrophils, and, to some extent, total leukocytes while preferentially increasing peripheral platelet counts in fully reconstituted mice. When analyzed 5 months posttransplant, tertiary MSCV-mIL-11 recipients had a significantly greater percentage of G418-resistant colony-forming cells in their bone marrow compared with control MSCV animals. Collectively, these data show that persistent stimulation of platelet production by IL-11 is not detrimental to stem cell repopulating ability; rather, they suggest that IL-11 expression in vivo may have resulted in enhanced maintenance of the most primitive hematopoietic stem cell compartment. The prolonged expression achieved by the MSCV retroviral vector, despite the presence of a selectable marker, contrasts with the frequent transcriptional extinction observed with other retroviral vectors carrying two genes. These findings have potentially important implications for clinical bone marrow transplantation and gene therapy of the hematopoietic system.

Clonal analysis of stably transduced human epidermal stem cells in culture.

We have transduced normal human keratinocytes with retroviral constructs expressing a bacterial beta-galactosidase (beta-gal) gene or a human interleukin-6 (hIL-6) cDNA under control of a long terminal repeat. Efficiency of gene transfer averaged approximately 50% and 95% of clonogenic keratinocytes for beta-gal and hIL-6, respectively. Both genes were stably integrated and expressed for more than 150 generations. Clonal analysis showed that both holoclones and their transient amplifying progeny expressed the transgene permanently. Southern blot analysis on isolated clones showed that many keratinocyte stem cells integrated multiple proviral copies in their genome and that the synthesis of the exogenous gene product in vitro was proportional to the number of proviral integrations. When cohesive epidermal sheets prepared from stem cells transduced with hIL-6 were grafted on athymic animals, the serum levels of hIL-6 were strictly proportional to the rate of secretion in vitro and therefore to the number of proviral integrations. The possibility of specifying the level of transgene expression and its permanence in a homogeneous clone of stem cell origin opens new perspectives in the long-term treatment of genetic disorders.

Proliferation of multipotent hematopoietic cells controlled by a truncated erythropoietin receptor transgene.

The long-term efficacy of gene therapy using bone marrow transplantation requires the engraftment of genetically altered totipotent hematopoietic stem cells (THSCs). Ex vivo expansion of corrected THSCs is one way to increase the efficiency of the procedure. Similarly, selective in vivo expansion of the therapeutic THSCs rather than the endogenous THSCs could favor the transplant. To test whether a conferred proliferative advantage gene can facilitate the in vitro and in vivo expansion of hematopoietic stem cells, we have generated transgenic mice expressing a truncated receptor for the growth factor erythropoietin. These mice are phenotypically normal, but when treated in vivo with exogenous erythropoietin they exhibit a marked increase in multipotent, clonogenic hematopoietic cells [colony-forming units in the spleen (CFU-S) and CFUs that give rise to granulocytes, erythroid cells, macrophages, and megakaryocytes within the same colony (CFU-GEMM)] in comparison with the wild-type mice. In addition, long-term in vitro culture of tEpoR transgenic bone marrow in the presence of erythropoietin induces exponential expansion of trilineage hematopoietic stem cells not seen with wild-type bone marrow. Thus, the truncated erythropoietin receptor gene shows promise as a means for obtaining cytokine-inducible hematopoietic stem cell proliferation to facilitate the direct targeting of THSCs and to provide a competitive repopulation advantage for transplanted therapeutic stem cells.

Gene-targeted deletion and replacement mutations of the T-cell receptor beta-chain enhancer: the role of enhancer elements in controlling V(D)J recombination accessibility.

To assess the role of transcriptional enhancers in regulating accessibility of the T-cell receptor beta-chain (TCRbeta) locus, we generated embryonic stem cell lines in which a single allelic copy of the endogenous TCRbeta enhancer (Ebeta) was either deleted or replaced with the immunoglobulin heavy-chain intronic enhancer. We assayed the effects of these mutations on activation of the TCRbeta locus in normal T- and B-lineage cells by RAG-2 (recombination-activating gene 2)-deficient blastocyst complementation. We found that Ebeta is required for rearrangement and germ-line transcription of the TCRbeta locus in T-lineage cells. In the absence of Ebeta, the heavy-chain intronic enhancer partially supported joining region beta-chain rearrangement in T- but not in B-lineage cells. However, ability of the heavy-chain intronic enhancer to induce rearrangements was blocked by linkage to an expressed neomycin-resistance gene (neo(r)). These results demonstrate a critical role for Ebeta in promoting accessibility of the TCRbeta locus and suggest that additional negative elements may cooperate to further modulate this process.

Cellular mechanisms for the formation of a soluble form derivative of T-cell receptor alpha chain by suppressor T cells.

Upon stimulation with anti-CD3, suppressor T-cell (Ts) hybridomas and homologous transfectants of T-cell receptor a (TCRalpha) cDNA in the T-cell hybridoma formed a 55-kDa TCRalpha chain derivative that bound both the monoclonal anti-TCRalpha chain and polyclonal antibodies against glycosylation inhibiting factor (GIF). The peptide is a subunit of antigen-specific suppressor T-cell factor (TsF), and is considered to be a posttranslationally-formed conjugate of TCRalpha chain with GIF peptide. The TCRalpha derivative is synthesized by the transfectant after stimulation with anti-CD3, and not derived from TCR present on the cell surface. Stimulation of the stable homologous transfectants with anti-CD3 induced translocation of the 13-kDa GIF peptide into endoplasmic reticulum (ER). When a helper Ts hybridoma or a stable transfectant of the same TCRalpha cDNA in a helper cell-derived TCRalpha- clone was stimulated with anti-CD3, translocation of GIF peptide was not detected, and these cells failed to secrete a TCRalpha derivative. However, further transfection of a chimeric cDNA encoding a procalcitonin-GIF fusion protein into the helper cell-derived stable transfectant of TCRalpha cDNA resulted in translocation of the GIF protein and formation of bioactive 55-kDa GIF. The results indicated that translocation of GIF peptide through ER is unique for Ts cells, and that this process is essential for the formation/secretion of the soluble form derivative of TCRalpha chain by T cells.

Long-term culture of lymphohematopoietic stem cells.

Pluripotent hematopoietic stem cells (PHSCs) show self-renewal and give rise to all blood cell types. The extremely low number of these cells in primary hematopoietic organs and the lack of culture systems that support proliferation of undifferentiated PHSCs have precluded the study of both the biology of these cells and their clinical application. We describe here cell lines and clones derived from PHSCs that were established from hematopoietic cells from the fetal liver or bone marrow of normal and p53-deficient mice with a combination of four growth factors. Most cell lines were Sca-1+, c-Kit+, PgP-1+, HSA+, and Lin- (B-220-, Joro 75-, 8C5-, F4/80-, CD4-, CD8-, CD3-, IgM-, and TER 119-negative) and expressed three new surface markers: Joro 177, Joro 184, and Joro 96. They did not synthesize RNA transcripts for several genes expressed at early stages of lymphocyte and myeloid/erythroid cell development. The clones were able to generate lymphoid, myeloid, and erythroid hematopoietic cells and to reconstitute the hematopoietic system of irradiated mice for a long time. The availability of lymphohematopoietic stem cell lines should facilitate the analysis of the molecular mechanisms that control self-renewal and differentiation and the development of efficient protocols for somatic gene therapy.

Genetic ablation of parietal cells in transgenic mice: a new model for analyzing cell lineage relationships in the gastric mucosa.

The gastric mucosa of mammalian stomach contains several differentiated cell types specialized for the secretion of acid, digestive enzymes, mucus, and hormones. Understanding whether each of these cell lineages is derived from a common stem cell has been a challenging problem. We have used a genetic approach to analyze the ontogeny of progenitor cells within mouse stomach. Herpes simplex virus 1 thymidine kinase was targeted to parietal cells within the gastric mucosa of transgenic mice, and parietal cells were ablated by treatment of animals with the antiherpetic drug ganciclovir. Ganciclovir treatment produced complete ablation of parietal cells, dissolution of gastric glands, and loss of chief and mucus-producing cells. Termination of drug treatment led to the reemergence of all major gastric epithelial cell types and restoration of glandular architecture. Our results imply the existence of a pluripotent stem cell for the gastric mucosa. Parietal cell ablation should provide a model for analyzing cell lineage relationships within the stomach as well as mechanisms underlying gastric injury and repair.

Embryonic stem cells express multiple Eph-subfamily receptor tyrosine kinases.

Eph and its homologues form the largest subfamily of receptor tyrosine kinases. Normal expression patterns of this subfamily indicate roles in differentiation and development, whereas their overexpression has been linked to oncogenesis. This study investigated the potential role of Eph-related molecules during very early embryonic development by examining their expression in embryonic stem (ES) cells and embryoid bodies differentiated from ES cells in vitro. By use of a strategy based on reverse transcriptase-mediated PCR, nine clones containing Eph-subfamily sequence were isolated from ES cells. Of these, eight were almost identical to one of four previously identified molecules (Sek, Nuk, Eck, and Mek4). However, one clone contained sequence from a novel Eph-subfamily member, which was termed embryonic stem-cell kinase or Esk. Northern analysis showed expression of Esk in ES cells, embryoid bodies, day 12 mouse embryos, and some tissues of the adult animal. Levels of expression were similar in ES cells and embryoid bodies. By comparison, Mek4 showed no significant transcription in the ES cell cultures by Northern analysis, whereas Eck displayed stronger signals in ES cells than in the embryoid bodies. These results suggest that Eph-subfamily molecules may play roles during the earliest phases of embryogenesis. Furthermore, the relative importance of different members of this subfamily appears to change as development proceeds.

Enhanced somatic mutation rates induced in stem cells of mice by low chronic exposure to ethylnitrosourea.

We have found that the somatic mutation rate at the Dlb-1 locus increases exponentially during low daily exposure to ethylnitrosourea over 4 months. This effect, enhanced mutagenesis, was not observed at a lacI transgene in the same tissue, although the two loci respond very similarly to acute doses. Since both mutations are neutral, the mutant frequency was expected to increase linearly with time in response to a constant mutagenic exposure, as it did for lacI. Enhanced mutagenesis does not result from an overall sensitization of the animals, since mice that had first been treated with a low daily dose for 90 days and then challenged with a large acute dose were not sensitized to the acute dose. Nor was the increased mutant frequency due to selection, since animals that were treated for 90 days and then left untreated for up to 60 days showed little change from the 90-day frequency. The effect is substantial: about 8 times as many Dlb-1 mutants were induced between 90 and 120 days as in the first 30 days. This resulted in a reverse dose rate effect such that 90 mg/kg induced more mutants when delivered at 1 mg/kg per day than at 3 mg/kg per day. We postulate that enhanced mutagenesis arises from increased stem cell proliferation and the preferential repair of transcribed genes. Enhanced mutagenesis may be important for risk evaluation, as the results show that chronic exposures can be more mutagenic than acute ones and raise the possibility of synergism between chemicals at low doses.

The murine stk gene product, a transmembrane protein tyrosine kinase, is a receptor for macrophage-stimulating protein.

Macrophage-stimulating protein (MSP) was originally identified as an inducer of murine resident peritoneal macrophage responsiveness to chemoattractants. We recently showed that the product of RON, a protein tyrosine kinase cloned from a human keratinocyte library, is the receptor for MSP. Similarity of murine stk to RON led us to determine if the stk gene product is the murine receptor for MSP. Radiolabeled MSP could bind to NIH 3T3 cells transfected with murine stk cDNA (3T3/stk). Binding was saturable and was inhibited by unlabeled MSP but not by structurally related proteins, including hepatocyte growth factor and plasminogen. Specific binding to STK was demonstrated by cross-linking of 125I-labeled MSP to membrane proteins of 3T3/stk cells, which resulted in a protein complex with a molecular mass of 220 kDa. This radiolabeled complex comprised 125I-MSP and STK, since it could be immunoprecipitated by antibodies to the STK beta chain. Binding of MSP to stk cDNA-transfected cells induced tyrosine phosphorylation of the 150-kDa STK beta chain within 1 min and caused increased motile activity. These results establish the murine stk gene product as a specific transmembrane protein tyrosine kinase receptor for MSP. Inasmuch as the stk cDNA was cloned from a hematopoietic stem cell, our data suggest that in addition to macrophages and keratinocytes, a cell in the hematopoietic lineage may also be a target for MSP.

High incidence of XXY and XYY males among the offspring of female chimeras from embryonic stem cells.

Injecting male embryonic stem cells into the blastocoel of female embryos occasionally produces female chimeras capable of transmitting the embryonic stem cell genome. In our experiments several embryonic stem cell-derived male offspring from female chimeras were observed to be infertile. Karyotypic analysis of these infertile animals revealed aneuploidy. We examined the karyotypes of an additional 14 offspring not selected for infertility (3 females and 11 males) that had received the embryonic stem cell genome from 5 transmitting female chimeras. The 3 females and 5 of the males had normal karyotypes. Six of the males exhibited nonmosaic aneuploidy, which included four XXY karyotypes, one XYY karyotype, and an X,i(Y) karyotype. The high incidence of XXY and XYY males supports previous evidence for aberrant pairing and segregation of X and Y chromosomes when they are present in oocytes.