We identified a novel nuclear protein, NF-|jiNR, that binds to multiple sites flanking the immunoglobulin heavy-chain enhancer. The expression of NF-|jiNR 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-|iNR 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-|jiNR correlate very well with cisacting negative regulatory elements of the heavy-chain enhancer defined previously. Indeed, when the segments bound by NF-^.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-|jiNR may function as a negative regulator of enhancer function. The observation that the segments bound by NF-ftNR 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.

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 ofmixed 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.

Mouse embryonic stem (ES) cells can differentiate in culture to late stages of many cell lineages. 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. 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.

Mouse embryonic stem cells (ES) were allowed to differentiate in a liquid culture system. After 23 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 uitro 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 andlor 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.0 1993 Wiley-Liss, Inc.

Mouse embryonic stem (ES) cells in culture can differentiate into late stages of many lineage-committed precursor cells. Under appropriate organ-culture conditions, ES cels differentiate into lymphoidlike cells at a stage equivalent to lymphoid cells found in fetal liver. These hematopoietic precursors are located in cup-shaped structures found in some embryoid bodies; we called such embryoid bodies “ES fetuses.” In this study, we have followed the maturation of hematopoietic cells after implantation of ES fetuses into nude mice for 3 weeks. ES-cell-derived lymphoid cells-pre-B cells, mature B cells, and mature T cells were found in all lymphoid organs. Interestingly, there was also an increase of T cells of host origin. Because native nude mouse lack thymus, these T cells might be educated by thymuslike epithelium generated from ES fetuses. Practical applications of this combined in vitro and in vivo system are discussed.

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 cells. In this review, we discuss the data on the development of lymphoid tissues, skin, gut, and other tissues originating from embryonic stem cells in vitro. We explore the prospects of using mouse embryonic stem cells for tissue engineering. QUASI-TOTIPOTENT VERSUS TOTIPOTENT L INES OF MOUSE EMBRYONIC STEM (ES) CELLS can be established from the inner cell mass of blastocysts.The ES cells may be easily manipulated, and specific genes may be replaced. They resemble germ cells in that they are "quasi-totipotent." That is, when ES cells are reintroduced in vivo, either by blastocyst injection or by chimeric aggregation, they are able to participate in all stages of embryogenesis in the uterus of the foster mother. The immediate offspring are chimeric, but animals genetically identical to the ES cells may be obtained by interbreeding. In this way, genetically manipulated ES cells can give rise to whole animals of the manipulated genotype. In culture, ES cells may develop into many cell types. It is often assumed that ES cells are also totipotent in vitro, but this has never been clearly proven. As the only really definitive proof would be the development of a living and functional "fetus" from ES cells by culturing in petri dishes, we will just have to live with the concept of quasi-totipotency.The genetic information governing the generation of an embryo resides in the genome of the ES cells, but that information must be read out in a regulated fashion. If all of embryogenesis is an autocrine process, and if embryogenesis can proceed without errors only when the microenvironment permits, then culture conditions, both physical and nutrient, during the differentiation of ES cells in culture must mimic the environment of the uterus of a pregnant female, but the concept of optimal culture conditions is itself wishful thinking, because the uterine environment constantly changes during the course of pregnancy.

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.