Normal Mouse Bone Marrow Research Articles

Reactivation of a hematopoietic endocrine program of pregnancy contributes to recovery from thrombocytopenia.

Regulation of blood platelet levels involves an array of cytokines, including the placental hormone PRL-like protein E (PLP-E). The PLP-E receptor is present on megakaryocytes in pregnant mice, nonpregnant female mice, and male mice. Other known megakaryocytic cytokines do not share the PLP-E receptor, and thus the presence of this receptor in nonpregnant animals suggests that PLP-E may be expressed in tissues other than the placenta. Consistent with this prediction, PLP-E is produced in thrombocytopenic mouse bone marrow, primarily in granulocytes, but not in normal mouse bone marrow. Serum from thrombocytopenic mice, purified thrombopoietin or IL-6, or pregnancy can induce bone marrow cell expression of PLP-E. The induction of PLP-E gene expression in response to thrombocytopenia is physiologically significant, as injection of PLP-E into thrombocytopenic mice restores normal platelet levels with no effect on granulocytes, erythrocytes, and total white blood cell counts. We conclude that inducible expression of PLP-E in bone marrow is part of the mechanism of recovery from thrombocytopenia. These results also suggest a more general concept: that the endocrine program of pregnancy, which in mammals has evolved to support the intrauterine growth and development of the fetus, can also be harnessed to respond to pathophysiology.

Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells.

We have derived from normal human, mouse, and rat postnatal bone marrow primitive, multipotent adult progenitor cells (MAPCs) that can differentiate into most mesodermal cells and neuroectodermal cells in vitro and into all embryonic lineages in vivo. Here, we show that MAPCs can also differentiate into hepatocyte-like cells in vitro. Human, mouse, and rat MAPCs, cultured on Matrigel with FGF-4 and HGF, differentiated into epithelioid cells that expressed hepatocyte nuclear factor-3beta (HNF-3beta), GATA4, cytokeratin 19 (CK19), transthyretin, and alpha-fetoprotein by day 7, and expressed CK18, HNF-4, and HNF-1alpha on days 14-28. Virtually all human, as well as a majority of rodent cells stained positive for albumin and CK18 on day 21; 5% (rodent) to 25% (human) cells were binucleated by day 21. These cells also acquired functional characteristics of hepatocytes: they secreted urea and albumin, had phenobarbital-inducible cytochrome p450, could take up LDL, and stored glycogen. MAPCs, which can be expanded in vitro and maintained in an undifferentiated state for more than 100 population doublings, can thus differentiate into cells with morphological, phenotypic, and functional characteristics of hepatocytes. MAPCs may therefore be an ideal cell for in vivo therapies for liver disorders or for use in bioartificial liver devices.

Age-Related Osteoporosis in Biglycan-Deficient Mice Is Related to Defects in Bone Marrow Stromal Cells

Biglycan (bgn) is an extracellular matrix proteoglycan that is enriched in bone and other skeletal connective tissues. Previously, we generated bgn-deficient mice and showed that they developed age-dependent osteopenia. To identify the cellular events that might contribute to this progressive osteoporosis, we measured the number of osteogenic precursors in the bone marrow of normal and mutant mice. The number of colonies, indicative of the colony-forming unit potential of fibroblasts (CFU-F), gradually decreased with age. By 24 weeks of age, colony formation in the bgn knockout (KO) mice was significantly more reduced than that in the wild type (wt) mice. This age-related reduction was consistent with the extensive osteopenia previously shown by X-ray analysis and histological examination of 24-week-old bgn KO mice. Because bgn has been shown previously to bind and regulate transforming growth factor beta (TGF-beta) activity, we also asked whether this growth factor would affect colony formation. TGF-beta treatment significantly increased the size of the wt colonies. In contrast, TGF-beta did not significantly influence the size of the bgn colonies. An increase in apoptosis in bgn-deficient bone marrow stromal cells (BMSCs) was observed also. The combination of decreased proliferation and increased apoptosis, if it occurred in vivo, would lead to a deficiency in the generation of mature osteoblasts and would be sufficient to account for the osteopenia developed in the bgn KO mice. The bgn KO mice also were defective in the synthesis of type I collagen messenger RNA (mRNA) and protein. This result supports the suggestion that the composition of the extracellular matrix may be regulated by specific matrix components including bgn.

Multipotential hematopoietic stem cells in the bone marrow

It has been generally held that human hematopoietic stem cells are lineage-negative CD34+ CD38−. However, murine hematopoietic stem cells were reported to be CD34−. We have characterized the surface phenotypes of murine hematopoietic stem cells by using a murine transplantation model. Our studies revealed that the majority of the stem cells in normal adult mice are CD34− while a minority (15%–20%) being CD34+. Our studies also revealed that stem cells that are activated by injection of 5-fluorouracil in vivo, exposure to cytokines in vitro, or mobilization by G-CSF are CD34+ and that CD34 expression is reversible. It has been reported that fetal murine hematopoietic stem cells are CD34+. Our studies revealed that stem cells of juvenile mice are CD34+ and that the developmental change from CD34+ to CD34− state takes place between 7 and 10 weeks of age. In adult mice, expression of CD38 by steady-state and activated stem cells was completely reciprocal of CD34 expression. Activated stem cells and the minority population of the stem cells in the normal mice are CD34+ CD38−. In contrast, the majority of stem cells in normal adult mice are CD34- CD38+. Recently, we studied CD38 expression by stem cells of neonatal and juvenile mice. Stem cells of newborn mice are CD38−. About half of the stem cells of 5-week-old mice are CD38+. Finally, our studies indicated that some of the CD34+ stem cells in the bone marrow of normal adult mice express lineage markers such as Mac-1 and CD4. These studies in a murine model clearly documented that expression of both CD34 and CD38 by stem cells is under developmental control and may be subject to changes induced by activation of the stem cells. In order to test whether or not these principles apply to human stem cells we tested surface phenotypes of human stem cells using two xenotransplantation techniques. Studies based on human/sheep xenograft model indicated that a significant portion of adult human long-term engrafting cells are CD34−. Similar to mouse stem cells expression of CD34 by human stem cells was reversible. Studies based on our newborn NOD/SCID/β-microglobulinnull mice indicated that human cord blood stem cells are CD34+ CD38−. These results appear to support the validity of studies of murine stem cells to provide insight into human stem cells.

Changing phenotypes of hematopoietic stem cells.

Identification of the surface phenotypes of hematopoietic stem cells is an important subject in both clinical stem cell transplantation and basic research in hematopoiesis. Transplantation of pure populations of stem cells should eliminate the occurrence of graft-vs-host disease (GVHD) in allogeneic transplantation and may reduce the recurrence rate of malignancies in autologous transplantation. Availability of highly enriched populations of stem cells should facilitate investigations in a number of areas such as in vitro expansion, cryogenic storage, and gene therapy using stem cells. An ultimate goal is to develop a method to quantitate the number of living stem cells based on their surface characteristics. During the last two decades, investigators identified a number of surface molecules, which, in combinations, were thought to provide exact definition of murine and human stem cells. Recent studies of murine stem cells, however, clearly demonstrated that expression of the surface antigens of stem cells is under the influence of developmental stages and the kinetic state of the stem cells. This review summarizes the concept of the changing phenotypes of hematopoietic stem cells.

HEMOPOIESIS-STIMULATING EFFECTS AND ENHANCED SURVIVAL OF IRRADIATED MICE AFTER PERORAL OR INTRAPERITONEAL ADMINISTRATION OF ULTRAFILTERED PIG LEUKOCYTE EXTRACT (UPLE, IMUNOR®)

ABSTRACTUltrafiltered pig leukocyte extract (UPLE, IMUNOR®, ImunomedicA, Ústí nad Labem, Czech Republic) administered perorally (p.o.) or intraperitoneally (i.p.) enhanced recovery of the pool of granulocyte-macrophage hemopoietic progenitor cells (GM-CFC) in the bone marrow of normal or sublethally irradiated mice and increased survival of mice exposed to a lethal radiation dose. In experiments in vitro, sera of mice treated with UPLE p.o. or i.p. induced GM-CFC colony formation in cultures of normal mouse bone marrow cells, i.e., produced colony-stimulating activity (CSA). UPLE alone did not induce GM-CFC colony growth, i.e., had no CSA. When UPLE alone or sera of mice administered UPLE p.o. or i.p. were added to bone marrow cultures containing suboptimal concentration of recombinant mouse interleukin-3 (rmIL-3), both UPLE and the sera increased the counts of GM-CFC colonies in comparison with cultures containing only rmIL-3, i.e., produced co-stimulating activity (CoSA). Based on the findings obtained in vitro, it can be hypothesized that the described CSA and CoSA of UPLE may play a role also under in vivo conditions; enhancement of the recovery of hemopoiesis suppressed by ionizing radiation may be due to co-operation of the stimulatory effects of UPLE with the action of cytokines endogenously produced in irradiated tissues.

Bone marrow stromal cells mediate androgenic suppression of B lymphocyte development

Castration of normal male mice induces expansion of the bone marrow B cell population, an effect that can be reversed by androgen replacement. We employed in vitro cultures and two in vivo models to investigate whether androgens exert these effects directly on marrow lymphoid precursors or whether actions on marrow stromal elements are required. Immature B cells from normal mouse bone marrow were not responsive to the suppressive effect of androgens unless they were cocultured with marrow stromal cells or with supernatants from androgen-treated stromal cells, suggesting that the androgen effects are exerted through marrow stromal elements by production of a diffusible mediator. Further experiments revealed that bone marrow stromal cells produced TGF-β in response to dihydrotestosterone (DHT), and neutralization of TGF-β in the DHT-treated stromal cells reversed the suppressive effects. The stromal cell requirement for androgen-mediated effects was confirmed in vivo by experiments using chimeric animals created by bone marrow transplantation in which androgen receptor expression was restricted to either the stromal or lymphoid cells of the bone marrow. Androgens only affected B cell development in chimeric mice with androgen-sensitive stromal cells. These experiments suggest that effects of androgens on developing B cells are mediated through androgen receptors in bone marrow stromal cells. TGF-β is a candidate mediator for these hormonal effects.

Bone marrow stromal cells mediate androgenic suppression of B lymphocyte development

Castration of normal male mice induces expansion of the bone marrow B cell population, an effect that can be reversed by androgen replacement. We employed in vitro cultures and two in vivo models to investigate whether androgens exert these effects directly on marrow lymphoid precursors or whether actions on marrow stromal elements are required. Immature B cells from normal mouse bone marrow were not responsive to the suppressive effect of androgens unless they were cocultured with marrow stromal cells or with supernatants from androgen-treated stromal cells, suggesting that the androgen effects are exerted through marrow stromal elements by production of a diffusible mediator. Further experiments revealed that bone marrow stromal cells produced TGF-beta in response to dihydrotestosterone (DHT), and neutralization of TGF-beta in the DHT-treated stromal cells reversed the suppressive effects. The stromal cell requirement for androgen-mediated effects was confirmed in vivo by experiments using chimeric animals created by bone marrow transplantation in which androgen receptor expression was restricted to either the stromal or lymphoid cells of the bone marrow. Androgens only affected B cell development in chimeric mice with androgen-sensitive stromal cells. These experiments suggest that effects of androgens on developing B cells are mediated through androgen receptors in bone marrow stromal cells. TGF-beta is a candidate mediator for these hormonal effects.

Somatic stem cell plasticity: current evidence and emerging concepts.

In the 19 th century, mammalian tissues were first described to be composed of cells, leading to the claim that cells originate exclusively from other cells (“omnis cellula a cellula”) formulated by Virchow and Schwann, respectively [1,2]. At the beginning of the 20 th century, the concept of tissue stem cells as the basis for tissue regeneration was introduced: analyzing the phylogeny of hematopoiesis in the bone marrow solely based on morphological observations, Pappenheim postulated the existence of an undifferentiated stem cell (“gemeinsame Stammzelle”) giving rise to the plethora of blood cells via an intermediate state of progenitor cells (Fig. 1, [3]). In the 1950s, several groups corroborated the existence of the hematopoietic stem cell in the bone marrow by showing hematopoietic recovery from transplanted bone marrow after irradiation damage [4–6]. Till and McCulloch later traced hematopoietic repopulation capacity to clonogenic cells establishing spleen colony-forming units [7]. Subsequently, the concept of tissue regeneration from a small population of resident tissue stem cells was generally accepted, was extended to nonhematopoietic tissues such as gut and skin [8], and still is our understanding of adult tissue regeneration today, enriched by an immense body of descriptive data. In parallel, the principle of directed cellular proliferation underlay the understanding of the early stages in embryogenesis and, together with the cellular movement, led to the discovery of morphogenesis via germ layers in the early embryo [9]. With emerging technologies, it was 33 and 3 years ago that stem cells with the capacity to differentiate into all tissues of the adult organism were functionally isolated from preimplantation embryos in mice and humans, respectively, and were called embryonic stem (ES) cells [10–13]. Although the concept of stem cells in embryogenesis and stem cells in adult tissue regeneration were initially pursued in conceptually separate approaches, they merged again with the successful cloning of a mammal from the nucleus of an adult tissue cell 4 years ago [14]. These experiments established that the nuclei of at least some adult cells were capable of being reprogrammed and spurred several groups to reevaluate the differentiation capacity of adult tissue stem cells, leading to a number of reports on somatic stem cell plasticity over the last 3 years. Here, we will review the current evidence for stem cell plasticity. Following the chronology of discoveries, we will start from the broadening developmental potential of bone marrow–derived stem cells leading to the differentiation capacities of stem cells from nonhematopoietic tissues. We will discuss some of the potential caveats to the current work, and finally will speculate about the potential underlying mechanisms of transdifferentiation.

Constitutive expression of macrophage-inflammatory protein 2 (MIP-2) mRNA in bone marrow gives rise to peripheral neutrophils with preformed MIP-2 protein.

Macrophage-inflammatory protein 2 (MIP-2) is a major CXC chemokine involved in the migration of polymorphonuclear neutrophils (PMNs) to sites of inflammation. Although cell culture experiments have identified different cell types that can produce MIP-2, the cellular sources in vivo are not clearly defined. By using immunohistochemical staining and analysis of chemokine mRNA expression, the present study aimed to localize cells producing MIP-2 in tissues of normal mice and mice challenged with Yersinia enterocolitica. The results showed a constitutive expression of MIP-2 mRNA in bone marrow (BM) of normal mice, but not in other organs such as spleen, lung, or liver. MIP-2 protein was found in all organs tested but it was exclusively associated with PMNs that stained positive with the cell surface marker Gr-1. Bacterial infection caused a 5-fold increase in the number of MIP-2-positive PMNs recruited to spleens concomitant with a strong increase of splenic MIP-2 mRNA. This correlated well with a 3-fold loss of MIP-2-producing cells in BM. Because MIP-2 mRNA expression in PMNs was increased after stimulation with TNF, the results indicate that newly recruited PMNs can supplement their MIP-2 content through TNF-stimulated transcription. Together, the data imply a constitutive production of MIP-2 by a subset of PMNs in BM and argue for the possibility of a rapid mobilization of MIP-2 through its storage in circulating PMNs.

CONSTITUTIVE EXPRESSION OF IL-6-LIKE CYTOKINES IN NORMAL BONE MARROW: IMPLICATIONS FOR PATHOPHYSIOLOGY OF MYELOMA

Myeloma is a neoplasm thought to “home” to bone marrow. However, evidence for bone-marrow-specific receptors or adhesion molecules expressed on myeloma cells is scanty. Initial myeloma expansion is thought to be due to IL-6 and/or related cytokines. Previous determinations of cytokine expression in bone marrow were performed on bone marrow stromal lines; these findings may not reflect the constitutive pattern of expression in situ. Intracytoplasmic staining for IL-6-like cytokines revealed constitutive expression of some factors in the bone marrow of normal mice, but not spleens. Spleens of myeloma-transplanted SCID mice expressed IL-6-like cytokines, indicative of induction of expression by myeloma. Some cytokines expressed in bone marrow induced myeloma proliferation in the presence of dexamethasone, demonstrating dependence of the myeloma on these cytokines. Our data imply that, rather than “homing” to bone marrow, myeloma cells proliferated within marrow cavities more than in other organs because of growth factors constitutively expressed by bone marrow cells. As myeloma progressed, we observed the induction of growth factor expression in spleen cells. Furthermore, because cytokines other than IL-6 may induce myeloma cell proliferation, therapy aimed at neutralizing IL-6 may not be the most effective method to treat this disease. These findings have implications for both the pathophysiology and therapy of multiple myeloma.

Regulation of cell survival during B lymphopoiesis: increased pre-B cell apoptosis in CD24-transgenic mouse bone marrow

CD24 (heat-stable antigen) is expressed in a developmentally regulated fashion by B cell precursors in mouse bone marrow (BM), but its role in B lymphopoiesis remains obscure. A slight overexpression of CD24 in transgenic (Tg) mice leads to depletion of B lymphoid cells in BM. The present study examines whether CD24 is involved in apoptotic selection of B lineage cells under normal microenvironmental conditions in vivo. Double immunofluorescence labeling and flow cytometry have been used to quantitate the apoptotic rates of phenotypically defined B cell populations in BM of CD24-Tg mice. Apoptosis of pre-B cells expressing cytoplasmic mu heavy chains of IgM but lacking surface (s)IgM was increased both ex vivo and in short-term culture, while the number of pre-B cells was halved compared to BM of normal mice. In contrast, B220+mu- pro-B cells and sIgM+ B lymphocytes showed no significant change in either apoptosis or number. The findings provide evidence that CD24 can play a role in vivo in modulating pre-B cell apoptosis, a quality control checkpoint in B cell development.

Connexin-43 gap junctions are involved in multiconnexin-expressing stromal support of hemopoietic progenitors and stem cells

AbstractGap junctions (GJs) provide for a unique system of intercellular communication (IC) allowing rapid transport of small molecules from cell to cell. GJs are formed by a large family of proteins named connexins (Cxs). Cx43 has been considered as the predominantly expressed Cx by hematopoietic-supporting stroma. To investigate the role of the Cx family in hemopoiesis, we analyzed the expression of 11 different Cx species in different stromal cell lines derived from murine bone marrow (BM) or fetal liver (FL). We found that up to 5 Cxs are expressed in FL stromal cells (Cx43, Cx45, Cx30.3, Cx31, and Cx31.1), whereas only Cx43, Cx45, and Cx31 were clearly detectable in BM stromal cells. In vivo, the Cx43-deficient 14.5- to 15-day FL cobblestone area–forming cells (CAFC)-week 1-4 and colony-forming unit contents were 26%-38% and 39%-47% lower than in their wild-type counterparts, respectively. The reintroduction of the Cx43 gene into Cx43-deficient FL stromal cells was able to restore their diminished IC to the level of the wild-type FL stromal cells. In addition, these Cx43-reintroduced stromal cells showed an increased support ability (3.7-fold) for CAFC-week 1 in normal mouse BM and 5-fold higher supportive ability for CAFC-week 4 in 5-fluorouracil-treated BM cells as compared with Cx43-deficient FL stromal cells. These findings suggest that stromal Cx43-mediated IC, although not responsible for all GJ-mediated IC of stromal cells, plays a role in the supportive ability for hemopoietic progenitors and stem cells.

Connexin-43 gap junctions are involved in multiconnexin-expressing stromal support of hemopoietic progenitors and stem cells

Gap junctions (GJs) provide for a unique system of intercellular communication (IC) allowing rapid transport of small molecules from cell to cell. GJs are formed by a large family of proteins named connexins (Cxs). Cx43 has been considered as the predominantly expressed Cx by hematopoietic-supporting stroma. To investigate the role of the Cx family in hemopoiesis, we analyzed the expression of 11 different Cx species in different stromal cell lines derived from murine bone marrow (BM) or fetal liver (FL). We found that up to 5 Cxs are expressed in FL stromal cells (Cx43, Cx45, Cx30.3, Cx31, and Cx31.1), whereas only Cx43, Cx45, and Cx31 were clearly detectable in BM stromal cells. In vivo, the Cx43-deficient 14.5- to 15-day FL cobblestone area–forming cells (CAFC)-week 1-4 and colony-forming unit contents were 26%-38% and 39%-47% lower than in their wild-type counterparts, respectively. The reintroduction of the Cx43 gene into Cx43-deficient FL stromal cells was able to restore their diminished IC to the level of the wild-type FL stromal cells. In addition, these Cx43-reintroduced stromal cells showed an increased support ability (3.7-fold) for CAFC-week 1 in normal mouse BM and 5-fold higher supportive ability for CAFC-week 4 in 5-fluorouracil-treated BM cells as compared with Cx43-deficient FL stromal cells. These findings suggest that stromal Cx43-mediated IC, although not responsible for all GJ-mediated IC of stromal cells, plays a role in the supportive ability for hemopoietic progenitors and stem cells.

A simple method for the preparation of quiescent, long-term repopulating stem cells

Adult murine hematopoietic stem cells are obtained from the bone marrow of normal mice or alternatively following treatment with a single injection of 5-fluorouracil (5-FU). These sources contain many developmentally diverse progenitor cells that are often problematic in having characteristics and behavior similar to stem cells. Current methods for removing these cells are time consuming, have low yields and the populations of primitive cells obtained are still heterogeneous. We have developed a simple, inexpensive method for preparing an ideal population of quiescent stem cells, in which virtually all of the non-stem cell progenitors have been removed. This population of cells does not contain any CFU-S8, CFU-S12 or CFC that proliferate in response to individual growth factors such as IL-3 and GM-CSF. However, a combination of growth factors used with a new lympho-myeloid CFC, stem cell assay reveals that >80% of the in vitro colony forming cells are lympho-myeloid stem cells. When FACS sorted, these cells have a Sca-1+Lin−Thy-1loc-kit− phenotype, just one of them being capable of generating as many as 2×108 progeny. Furthermore, the cells are able to fully repopulate the hematopoietic system and maintain the long-term survival of irradiated mice, but only when co-injected with other cells that have short-term repopulating, radio-protective ability. The preparation of this cell population is based on the principle that stem cells proliferate less frequently than other progenitors. While the traditional single injection of 5-FU removes some of the non-stem cell progenitors, not all are in cycle during this short period of exposure. However, multiple i.p. injections of 5-FU (200 mg/kg), each separated by 24 hr results in a cell population virtually completely ablated of non-stem cell progenitors, yet still containing many of the quiescent lympho-myeloid stem cells.

Functional conservation of platelet glycoprotein V promoter between mouse and human megakaryocytes

Objective In an attempt to clarify the megakaryo-specific regulatory mechanism of GPV gene transcription, we characterized the 5′-flanking region of the mouse GPV gene. Materials and Methods The promotor activity of a −481/+22 5′-fragment of the mouse GPV gene was examined in normal mouse bone marrow cells (BMC) and various human cell lines using two distinct reporter gene assay systems, luciferase and green fluorescence protein (GFP). Results When a DNA construct consisting of this fragment and a GFP reporter gene were transiently expressed in thrombopoietin-supported mouse BMC culture, GFP was identified only in megakaryocytes. The same construct expressed high levels of GFP in the human megakaryocytic Dami line. When assessed by dual luciferase assay, the full −481/+22 fragment could drive variable promoter activity in human as well as mouse megakaryocytic lines but did not work in non-megakaryocytic cells. Sufficient transcriptional activation of this fragment was restricted to the cells expressing apparent GPV mRNA. A deletion and point mutation study indicated that GATA and Ets motifs, typical cis-acting elements for platelet-specific genes, located of −75 and −46, respectively, were essential for promoter function. Conclusion The GPV promoter has the general characteristics found in platelet-specific genes, and the mechanism for megakaryocyte-specific, maturation-dependent regulation of GPV gene transcription is highly conserved between mouse and human. Analysis of GPV transcription mechanism utilizing human lines as well as BMC should provide new information on the final maturational process of megakaryocytes.

Age-related changes in activation of telomerase in the bone marrow of normal and thymectomized mice.

Age-related and thymus-dependent regulation of telomerase activity was studied in the bone marrow of normal (physiological aging) and thymectomized (experimental aging) mice. There was no strong correlation between the age and telomerase activity in bone marrow cells of normal mice. We observed only small individual differences in telomerase activity. Thymectomy 2-fold increased telomerase expression in young (2-month-old) and old (24-month-old) animals. Individual differences in telomerase activity in the bone marrow of thymectomized mice were more pronounced and did not depend on the age. The role of the thymus in cell aging is discussed.

Identification and characterization of a bipotent (erythroid and megakaryocytic) cell precursor from the spleen of phenylhydrazine-treated mice

AbstractWe have identified a cell population expressing erythroid (TER-119) and megakaryocyte (4A5) markers in the bone marrow of normal mice. This population is present at high frequency in the marrows and in the spleens involved in the erythroid expansion that occurs in mice recovering from phenylhydrazine (PHZ)-induced hemolytic anemia. TER-119+/4A5+ cells were isolated from the spleen of PHZ-treated animals and were found to be blast-like benzidine-negative cells that generate erythroid and megakaryocytic cells within 24-48 hours of culture in the presence of erythropoietin (EPO) or thrombopoietin (TPO). TER-119+/4A5+ cells represent a late bipotent erythroid and megakaryocytic cell precursors that may exert an important role in the recovery from PHZ-induced anemia.

Identification and characterization of a bipotent (erythroid and megakaryocytic) cell precursor from the spleen of phenylhydrazine-treated mice

We have identified a cell population expressing erythroid (TER-119) and megakaryocyte (4A5) markers in the bone marrow of normal mice. This population is present at high frequency in the marrows and in the spleens involved in the erythroid expansion that occurs in mice recovering from phenylhydrazine (PHZ)-induced hemolytic anemia. TER-119+/4A5+ cells were isolated from the spleen of PHZ-treated animals and were found to be blast-like benzidine-negative cells that generate erythroid and megakaryocytic cells within 24-48 hours of culture in the presence of erythropoietin (EPO) or thrombopoietin (TPO). TER-119+/4A5+ cells represent a late bipotent erythroid and megakaryocytic cell precursors that may exert an important role in the recovery from PHZ-induced anemia.

Upregulation of telomerase activity by X-irradiation in mouse leukaemia cells is independent of Tert, Terc, Tnks and Myc transcription.

X-irradiation of two mouse myeloid leukaemia cell lines was found to lead to increased telomerase activities. Maximal increases in activity at 24 h post-irradiation were approximately three times control unirradiated cell levels. These maxima were reached at between 3-5 Gy depending upon cell line. Peak activity was reached at 8h, remained elevated to 24 h and returned to control levels by 48 h. In contrast, X-irradiation did not activate telomerase in a telomerase-negative human fibroblast line, while in cultured normal mouse bone marrow cells irradiation appeared to reduce activities. No simple relationship between radiation-induced increases in telomerase activity in the myeloid leukaemia lines and the proportions of cells in the S or M phases of the cell cycle was apparent. Radiation-induced increases in activity were significantly reduced by inhibitors of transcription (actinomycin D, alpha-amanatin) and protein synthesis (cycloheximide). These data are consistent with two possibilities: (i) X-irradiation leads to increased transcription and/or translation of a component of telomerase, thus increasing activities; or (ii) X-irradiation induces the transcription of a positive regulator of telomerase activity. Northern blot analysis did not indicate that transcription of mTert, the catalytic subunit of telomerase, or mTerc, the RNA component, was elevated after irradiation. Similarly, no significant changes in the expression of Myc or Tnks, the tankyrase gene, two suspected telomerase regulators, were detected. These data are therefore consistent with the induction by X-irradiation of a positive regulator of telomerase activity other than Tnks or Myc or the core protein and RNA components of the enzyme.