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Based on our work in the past 10 years, we have concluded that mouse embryonic stem cells are "quasi-totipotent" in vitro; that is, they resemble and may even possess the full totipotency of germ c...

Mouse embryonic stem (ES) cells in culture can differentiate into late stages of many lineage-committed precursors. Under appropriate organ culture conditions, ES cells differentiate into lymphoid-like cells at an embryonic stage equivalent to day 10–14. These hematopoietic precursors are located in cup-shaped structures found in some embryoid bodies; we called such embryoid bodies "ES fetuses." We have followed the maturation of hematopoietic cells after implantation of ES fetuses into nude mice and SCID mice for 3 weeks. Nude mice seem to be better hosts than SCID mice for maturation of lymphoid cells. In chimeric nude mice, mature CD4+ and CD8+ T cells were found in the spleen. As nude mice lack a thymus, these T cells might have been educated by thymus-like epithelium generated from ES fetuses endogenously. Here we report finding organized epithelial structures resembling thymic cortex and medulla in the implanted ES fetuses. Using immunofluorescence and peroxidase staining, with flow cytometry, light...

Mouse embryonic stem cells (ES) were allowed to differentiate in a liquid culture system. After 2–3 weeks, complex cystic embryoid bodies developed. These bodies were composed of several structures identified as cardiac muscle and yolk sac blood islands as well as cupshape compartments containing a mixed population of hematopoietic stem cells. When these cystic embryoid bodies were implanted into adult mice, either subcutaneously or under the kidney capsule, they developed into various tissues. These included bone, blood vessels, cardiac muscle, nerves, and skin with hair follicles. In addition, highly differentiated, complicated tissues resembling intestinal epithelium with mucus glands or salivary glandular tissue were derived. The ES tissues from these in vitro developed embryoid bodies developed quickly within 2 to 3 weeks of implantation. This is in contrast to a minimal of 6 weeks for teratocarcinomas derived from embryonic carcinoma cells and/or the direct implantation of undifferentiated embryonic stem cells. Moreover, we found that there are different types of tissue developed upon different sites of implantation. The data suggest a local environment and/or growth factors are influential for ES tissue development. This system provides a possible means to purify and identify stem cells that give rise to specific tissues, and to study the factors regulating the commitment of these stem cells. © 1993 Wiley-Liss, Inc.

Mouse embryonic stem (ES) cells can differentiate in culture to late stages of many cell lineages. I have found culture conditions that are favorable for development in vitro of ES cells into hematopoietic cells at a stage equivalent to day 11-14 of fetal liver development. I describe here: (1) the growth conditions necessary for maintenance of ES cells in an undifferentiated state, and the conditions that allow differentiation of cystic embryoid bodies that contain precursors of most hematopoietic cell lineages, including lymphoid cells; (2) the development of lymphoid vessels from ES fetuses in vivo; (3) the characterization of lymphoid, erythroid, megakaryoid, and myeloid cells from ES fetuses; and (4) the cloning of cell lines representing lymphoid, myeloid lineage cells from differentiated ES cells.

We report here a partial characterization of a “tet-on” glia O2A precursor cell line established from the reverse tetracycline-transactivator (rtTA)-SV40 T antigen (Tag) double transgenic mice. In culture, withdrawal of doxycycline prevents proliferation and the cell line undergoes apoptosis. Importantly, differentiation into type-2-astrocytes and oligodendrocytes can be induced when the cell line is cultured, in the absence of doxycycline, and with epithelial stem cell lines secreting hIL3 or hIL6. In contrast, no maturation into progeny was observed when a hCNTF-secreting cell line was used as the co-culture partner under the same condition. In order to address the question of whether the morphological distinct cells—spindle and stellar shaped cells are of a similar or different cell types, we have performed cell size analysis of these cells by FACS and electro-physiology measurement by the patch clamping technique. They are of a similar cell size, but posses distinct electrophysiological properties—spindle cells are less mature than the stellar cells. These tet-on glia O2A precursor cells were implanted to sites of transected sciatic nerve of adult mice and kept in the precursor stage by feeding mice with doxycycline containing drinking water. The toe movement of injured foot was measured every 3 weeks and the electrophysiological property of motor neuron was determined three months after the operation. Preliminary data have shown that these tet-on glia precursor cells are not toxic to the implanted hosts and can enhance the recovery of damaged motor nerves.

Background Stem cells, having the property of self renewal, offer the promise of lifelong repair of damaged tissue. However, somatic tissue-committed primary stem cells are rare and difficult to expand in vitro. Genetically modified stem-like cells with the ability to expand conditionally provide a valuable tool with which to study stem cell biology, especially the cellular events of proliferation and differentiation. In addition, stem cells may be appropriate candidates for therapeutic applications. Methods Double transgenic mice possesing SV40 T antigen (Tag) under the control of the reverse tetracycline-transactivator (rtTA) were used to establish cell lines. One brain cell line was partially characterized by DNA sequencing, morphology, antigen expression using flow cytometry, confocal microscopy, and electrophysiology using the patch clamp technique. Cell cycle analysis was performed using propidium iodide staining; cell viability and H3-thymidine incorporation assays. The ability of this cell line to differentiate was assessed by confocal microscopy following co-culture with stem cells secreting cytokines. Results We report here the establishment and partial characterization of a cell line derived from the brain tissue of rtTA-SV40 Tag transgenic mice. Analysis of the morphology and antigen markers has shown that this cell line mimics some aspects of primary glial precursors. The results of electrophysiology are consistent with this and suggest that the cell line is derived from O2A glial precursor cells. Cell cycle progression of this cell line is doxycycline-dependent. In the absence of doxycycline, cells become apoptotic. Differentiation into mature type 2 astrocytes and (precursor) oligodendrocytes can be induced upon withdrawal of doxycycline and addition of epithelial stem cells secreting cytokine, such as hIL3 (human Interleukine 3) or hIL6 to the culture. In contrast, co-culturing with hCNTF (human Ciliary NeuroTrophic Factor)-secreting epithelial stem cells did not induce them to mature into progeny cell types. Conclusion The differentiation of this O2A glial precursor line does not occur automatically in culture. Additional external help is required from the cell-based delivery of appropriate transgenic cytokines. Withdrawal of doxycycline from the culture medium removes the proliferation signals and induces a fatal outcome. Copyright © 2001 John Wiley & Sons, Ltd.

For researchers interested in cell-based therapy, it is a great challenge to learn how to expand lymphoid cells and their precursor cells ex vivo under defined conditions. Lymphocytes are defined by their cell surface receptor – BCR (B Cell Receptor, or immunoglobulin, Ig) for B cells and TCR (T Cell Receptor) for T cells. Precursor B cells bear a pre-B receptor, which contains lambda 5 in mice (‘physi' in humans), and precursor T cells bear a pre-TCR-alpha. No biochemically and genetically defined surface receptors for the progenitors of lymphoid precursors have been reported. It is not entirely clear how committed stem cells differentiate into lymphoid cells, which then mature into effector cells. Many theories have been postulated. Differentiation and maturation involve both antigen-independent and antigen-dependent processes. For the first process, I will explore the sequential commitment model. For my treatment of the second process, I am indebted to many colleagues who helped me to summarize current views.

BrdU/Hoechst-EB bivariate flow cytometry has a wide application in the study of factors controlling cell cycle for asynchronous cells such as embryonic stem cells (ES), and for synchronous cells such as stimulated resting B cells (Bo). The technique allows one to calculate the average cell cycle duration time. ES cells are found to cycle every 8-10 h, and most B cells are 11-12 h, but there is a small subset of B cells with a cycle time of only 6-7 h. Using this technique, we also study the roles of different T lymphocytes on B cell activation when B cells are stimulated with anti-IgM antibodies (commonly used, anti-mu). Exposure to anti-mu recruits small B cells into the cell cycle, but arrests them in the G1 phase of the second cycle. Interleukin (IL) 4 is a costimulator of anti-mu. In addition, IL-4 is an S-phase progression factor. Contrary to that seen when B cells are stimulated by other mitogens, very few cells are in the G2 compartments after anti-mu plus IL-4 stimulation. This phenomenon is reminiscent of embryonic cells. Our findings provide strong evidence to propose that there are two restriction points for B cell activation: at the transition from G0 to G1 and at the transition from G1 to S phase.

Mouse embryonic stem (ES) cells have the potential to differentiate into embryoid bodies in vitro and mimic normal embryonic development. The "ES fetus" is a specific development at a late stage seen under our culture conditions. We have established several mixed populations from ES fetuses by using combinations of retroviruses carrying different oncogenes (v-abl, v-raf, c-myc), interleukins 2 and 3, and Con A. Six groups of mixed populations were characterized by immunophenotyping. For some groups, transfer of cells into sublethally irradiated mice resulted in the development of macrophages, mature T and B lymphocytes, and plasma cells of donor origin. Thus, these mixed populations may contain immortalized precursors of hematopoietic lineages. These mixed populations should be valuable for defining hematopoietic stem cells and their committed progenitors.

Stimulation of small, resting, splenic B cells with bacterial lipopolysaccharide (LPS) induces proliferation, differentiation to plasma cell formation, and the expression of immunoglobulin heavy chain (IgH). When this is combined with agents which crosslink surface Ig, differentiation and the induction of surface immunoglobulin are suppressed even though proliferation proceeds. We find that anti-mu antibodies suppresses Ig gene expression of transfected mu constructs, even if either the membrane or secretory segments have been deleted. We examined the effects of anti-mu treatment on the IgH enhancer (IgHE) attached to a heterologous test gene (CAT). Indeed the IgH enhancer alone was subject to anti-mu suppression, while the SV40 enhancer was insensitive. To determine what was responsible for suppression of enhancer function by anti-mu we examined nuclear extracts from stimulated splenic B cells for the presence of sequence-specific DNA binding activities to various sites within the enhancer. We found two specific differences--an induction in mu E5 binding activity, and a reduction in octamer transcription factor 2 (OTF2) binding activity, after anti-mu treatment. Analysis of these cells by in situ immunofluorescence with anti-OTF2 antibodies suggests that the nuclear localization of OTF2 in anti-mu treated cells may change, as well as its absolute level.

Activation of resting mouse B cells with anti-mu chain antibodies (anti-mu) leads to cell proliferation. We have investigated the effect of recombinant T cell interleukins (IL 2 to IL 6) on such anti-mu-induced proliferation. No proliferative response was detected when IL 2, IL 3 and IL 6, either alone or in combination with anti-mu, were studied. Furthermore, neither IL 4 nor IL 5 could induce proliferation when added alone to B cell cultures. However, when combined with anti-mu, IL 4 as well as IL 5 stimulated cell growth. Analysis by 5-bromo-2'-deoxyuridine/Hoechst 33258 flow cytometry revealed distinct effects of IL 4 and IL 5 on B cell growth. In the presence of anti-mu, both IL 4 and IL 5 co-stimulated unfractionated splenic B cells. However, when B cells were separated into subpopulations by density, IL 4 proved to be a cell cycle progression factor, stimulating the majority of resting B cells to enter the cell cycle. In contrast, IL 5 had little effect on the resting fraction of B cells. Rather, IL 5 acted as a co-competence factor, stimulating predominantly low-density B cells. Following exposure of anti-mu alone, most B cells accumulated in the G1 of the second cycle. Upon addition of IL 4, the cells acquired the ability to progress into the next S phase compartment. Contrary to what is seen when B cells are stimulated by other mitogens, very few cells are in the G2 compartments after anti-mu plus IL 4 stimulation. This phenomenon was not due to a differential cell cycle progression rate. Our findings provide an analytical basis for fractionating cell-cycle-compartment-specific B cells for their molecular study.

Publisher Summary This chapter discusses a cell-free system for polyadenylation using mouse B-cell extracts. The development of cell-free systems to study the initiation and regulation of transcription and the RNA processing reactions—splicing and polyadenylation—represents a major advance in the biochemical analysis of reactions. The chapter describes a detailed procedure for in vitro polyadenylation using cell-free protein extracts prepared from mouse cells of lymphoid origin. This system was primarily developed to study the posttranscriptional regulation of the mouse immunoglobulin M (IgM) locus. Polyadenylation of pre-RNA requires two independent steps. The first step is an endonucleolytic cleavage of the pre-RNA at a unique phosphate bond called the poly(A) site. After the cleavage approximately 200 adenosine residues are added to the 3´-OH of the upstream cleavage product. Sequences located both upstream and downstream of the poly(A) site are required for the reaction. The development of the in vitro polyadenylation system based on HeLa cell nuclear extract prompted several laboratories to study the reaction at the biochemical level.

Publisher Summary The chapter discusses in vitro transcriptional rate assay for lymphoid cells. In this assay, isolated nuclei are incubated briefly with [32P] UTP, and the incorporation into specific growing RNA chains is determined by hybridization to a selected set of immobilized DNA fragments. The initiation of new RNA chains is negligible, and chain elongation is only a few hundred nucleotides, so that the assay effectively measures the relative RNA polymerase loading of the various DNA segments. The nuclear run-on assay measures the relative RNA polymerase loading of various DNA segments. There are critical conditions that need to be met for such measurements to truly reflect the transcriptional activity. The genomic DNA or cDNA fragments used for the hybridization should not contain highly repetitive sequences that might hybridize with RNA transcribed elsewhere. The hybridizing sequences in the Southern blots should be in excess of those in the RNA to ensure that the amount of [32P]-labeled RNA hybridized is proportional to the RNA input. The state of cultured cells and the quality of nuclei preparation are critical to obtain good substrates for the elongation reaction.

Most techniques of flow cytometric cell cycle analysis are not capable of distinguishing the number of rounds of DNA synthesis that a cell has undergone since the start of an experiment. Continuous labeling with 5-bromodeoxyuridine (Brd-Urd) offers such a potential. We illustrate here that the bivariate analysis of non-BrdUrd-quenched ethidium bromide vs. BrdUrd-quenched Hoechst 33258 fluorescence offers a high degree of resolution that enhances the analytical power of the technique, and that this approach can be applied to the analysis of a broad range of human and murine primary cells and established cell lines.

Polyclonal stimulation of resting B cells with anti-antigen receptor antibodies [anti-IgM mu chain antibody (anti-mu)] or with bacterial lipopolysaccharide (LPS) stimulates a proportion of B cells to proliferate. Exposure of resting B cells to both LPS and anti-mu activates a larger population of resting B cells than either alone, suggesting a synergistic effect of these two stimuli. Although recombinant interferon (rIFN) either alone or in combination with anti-mu has no apparent proliferative activity (as measured by [3H]thymidine incorporation), application of 5-bromo-2'-deoxyuridine/Hoechst 33258 dye flow cytometry reveals a distinct effect of rIFN on B cell growth. In the presence of anti-mu plus LPS, rIFN causes the majority of B cells to enter the cell cycle (CC), but a subset of B cells remains in the resting stage. Another subset of B cells has extremely rapid CC transit times, with a CC duration of less than 10 h. These studies show that both anti-mu and LPS are competence factors (which move cells from the G0 phase to the G1 phase). LPS acts also as a CC progression factor, while rIFN is a CC potentiating factor.

We identified a novel nuclear protein, NF-mu NR, that binds to multiple sites flanking the immunoglobulin heavy-chain enhancer. The expression of NF-mu NR shows a unique developmental pattern; the activity is present in all cells representing early stages of B-cell development, but is absent from more mature cells that express a high level of immunoglobulin heavy chains. NF-mu NR also is present in most cell lines outside of the B-cell lineage (e.g., T cells, macrophages, and fibroblasts). The binding sites for NF-mu NR correlate very well with cis-acting negative regulatory elements of the heavy-chain enhancer defined previously. Indeed, when the segments bound by NF-mu NR are deleted from the enhancer, it is now found to function as a positive transcription element in T cells and macrophages. Taken together, these results suggest that NF-mu NR may function as a negative regulator of enhancer function. The observation that the segments bound by NF-mu NR correspond to the segments bound to the nuclear matrix suggests an intriguing model not only of how enhancers might function but also of how negative regulation might occur.

Over an average period of seven years 2,900 cases of benign breast lesions diagnosed by biopsy between 1948 and 1973 in the Department of Pathology, Kaiser Foundation Hospital, Oakland, were followed for breast cancer development. When classified according to traditional diagnostic categories, the cancer incidence per 1,000 person-years varies between 2.7 and 7.9 and appears to be elevated in comparison to expectations obtained from the Third National Cancer Survey, San Francisco Bay Area. Two thousand four hundred biopsies were also scored by the Black-Chabon method. There is an upward trend in the breast cancer incidence as the atypia score rises, a finding which confirms conclusions from a retrospective case-control study by Black et al.1