Humanizing bone marrow in immune-deficient mice.

Humanized mice are useful for studying human hematopoietic stem cells (HSCs) and their niche. In particular, clonal study of human HSC enables precise comparison of in vivo behavior between murine and human HSCs. A single HSC is able to reconstitute hematopoiesis even after serial transplantations in mice. While the life span of somatic cells is over that of individual in mice, this is not the case in humans. Clonal studies of human HSCs clearly demonstrated their aging in hosts. Since murine studies have demonstrated that HSCs are protected from aging by their niche in bone marrow, the humanizing niche model will reveal the precise mechanism by which human HSCs are protected from exhaustion in vivo. Direct transplantation of human mesenchymal stem cells into mouse bone marrow results in reconstitution of the functional human hematopoietic microenvironment comprised of pericytes, myofibroblasts, reticular cells, osteocytes in bone, bone-lining osteoblasts, and endothelial cells. These humanized mouse models are essential for testing whether the insights on hematopoiesis from mouse studies are applicable to humans before clinical application.

Osteoclast development from hematopoietic stem cells: apparent divergence of the osteoclast lineage prior to macrophage commitment.

To further clarify the progression of osteoclast development, the relationship of clonogenic osteoclast progenitors (CFU-O) to macrophage or more primitive progenitors was examined. Serum-free culture supernatant of a tumor clone (CESJ) was used as a source of an osteoclast colony stimulating factor (O-CSF). CFU-O-derived colonies were identified by their characteristic positive staining for tartrate resistant acid phosphatase (TRAPase). The effect of macrophage colony stimulating factor (M-CSF) and stem cell factor (SCF) on osteoclast progenitors was examined by pre-culturing mouse bone marrow (BM) cells in agar medium containing M-CSF or SCF and overlaying CESJ medium 0-7 days later. The number of TRAPase+ colonies decreased while TRAP- macrophage colonies increased in M-CSF pre-cultures as overlays of CESJ medium were delayed. On the other hand, TRAPase+ and mixed colonies persisted in SCF pre-cultures with CESJ medium overlays. Conversely, all colonies were TRAPase+ and no macrophage colonies developed in O-CSF pre-cultures overlaid with M-CSF. CFU-O, but not CFU-M, survived 7 days without exogenous CSFs in agar medium. In fractionated BM, the majority (> 99%) of CFU-O were in the c-kit positive population; however, a specific antibody to SCF did not affect O-CSF-induced TRAPase+ colony formation, suggesting the proliferation and differentiation of osteoclast progenitors are independent of c-kit-SCF interactions. These studies provide further experimental evidence to support the concept that O-CSF acts on progenitors in earlier stages of development, supporting their differentiation into the osteoclast lineage prior to macrophage commitment.

Relative roles of osteoclast colony-stimulating factor and macrophage colony-stimulating factor in the course of osteoclast development.

Although recent studies have shown that osteopetrotic (op/op) mice lack macrophage colony-stimulating factor (M-CSF or CSF-1), the precise role of M-CSF in the development of immature osteoclasts remains unknown. Using a recently discovered osteoclast-specific colony-stimulating factor (O-CSF) and in vitro long-term bone marrow culture systems, we investigated the ability of op/op and control marrow stromal cells to support the production of O-CSF-responsive clonogenic osteoclast progenitors (colony-forming unit-osteoclast [CFU-O]) from inoculated normal stem cells. Remarkably, op/op stromal cell cultures produced five times as many nonadherent cells as control cultures throughout the experimental period of 14 weeks; an average of 37% of these cells were nonviable compared with 8% in control cultures. Significantly higher numbers of CFU-O were found in op/op cultures than in control cultures; the CFU-O in op/op and control cultures were proliferating at a similar rate. Higher numbers of calcitonin receptor-bearing cells were found when harvested cells from op/op flasks were cultured with 1,25(OH)2D3. These studies clearly show that op/op marrow stromal cells can support the differentiation and proliferation of osteoclast progenitors from inoculated stem cells and provide the first experimental evidence that M-CSF is not essential for the early stages of osteoclast development. We hypothesize that while O-CSF supports proliferation of osteoclast progenitors, M-CSF plays a role in the later development and maturation of the progenitor as well as in the prevention of cell death.

Absence of a CD34- hematopoietic precursor population in recipients of CD34+ stem cell transplantation.

The purified CD34(+) cell fraction has been used for hematopoietic stem cell transplantation since they were demonstrated to have long-term reconstituting ability. Therefore, the potential effects of CD34(-) stem cells on the clinical course have been a major concern in recipients of CD34(+)-selected transplantation. To address this concern, we used an in vitro assay to determine whether transplant recipients have CD34(-)precursor population. Lin(-)CD34(-) cells were isolated from bone marrow cells in 11 transplant recipients including four CD34-selected transplantations, six standard bone marrow transplantations, and one T cell-depleted marrow transplantation. The frequency of the Lin(-)CD34(-) population in four CD34-enriched transplantation recipients was not different from those of normal donors or recipients of other modes of transplantation: 0.96 +/- 1.01% (mean +/- s.d., n = 4), 0.45 +/- 0.16% (n = 6), and 0.66 +/- 0.59% (n = 7), respectively. However, the Lin(-)CD34(-)population obtained from the recipients of CD34-enriched transplantation acquired neither CD34 expression nor colony-forming activity after 7 days of culture, whereas the cells from all the normal individuals and standard BMT recipients were able to differentiate into CD34(+) cells accompanied by the emergence of colony-forming activity.We conclude that recipients of CD34-enriched transplantation appear to have defects in their CD34(-) precursor population. The clinical significance of these defects will be determined in a life-long follow-up of these patients.

Contributionof Z-line constituents to the formation of the contraction bands of chicken myofibrils on addition of Mg2+-ATP.

1. Z-line constituents contributing to the formation of the contraction bands in chicken myofibrils were investigated by using the rabbit calcium-activated factor (CAF). 2. CAF digestion hampered the formation of the contraction bands and increased the amount of soluble proteins which are apparently derived from the Z-line. Among several proteins released by CAF digestion, a 95,000 dalton component, which corresponds to alpha-actinin, was predominant, followed by a 220,000 dalton component and several other proteins. 3. An apparent inverse relationship between the formation of the contraction bands and the release of Z-line proteins was observed. It is concluded that the presence of Z-line, including at least the two components mentioned above, is essential for the formation of the contraction bands.

Collaborative work to evaluate toxicity on male reproductive organs by repeated dose studies in rats 4). Fadrozole hydrochloride: an oral 2/4-week male reproductive organ toxicity study.

Doses of 0, 30 and 60 mg/kg/day of fadrozole hydrochloride (Afema: non-steroidal aromatase inhibitor, antitumor agent) were given perorally by gavage to HanIbm WIST male rats from 6 or 8 weeks of age for 2 weeks, and from 6 weeks of age for 4 weeks. In all treatment groups, reduced weights of seminal vesicle, prostate and epididymis, and degeneration/necrosis of the pachytene spermatocytes in stages VII or VIII seminiferous tubules, were dose-relatedly observed. Effects could also be assessed quantitatively by staging analysis with the result of a reduction in the numbers of stage VII pachytene spermatocytes at 30 and 60 mg/kg/day. Epididymal sperm examination revealed no treatment-related changes in any groups. The effects of 4-week treatment on male reproductive organs were similar to those of 2-week treatment at the same dose levels, except for the weights of seminal vesicle and prostate, which were more reduced by 4-week treatment than by 2-week treatment. There was no notable difference in detectability of toxicity in male reproductive organs between 2-week treatment from 6 weeks of age and 2-week treatment from 8 weeks of age. It was concluded that the changes observed in the rat male reproductive organs following 4 weeks of treatment with fadrozole hydrochloride could be detected also with 2 weeks of treatment.

[Prediction of bone marrow function by the appearance rate of RNA-rich reticulocytes during the chemotherapy for hematologic malignancy].

Percentage of RNA-rich (High fluorescence Ratio, HFR), RNA-middle (Middle Fluorescence Ratio, MFR) and RNA-low (Low Fluorescence Ratio, LFR) reticulocytes was estimated by Sysmex R-1000 reticulocyte counter during the chemotherapy of 27 cases with acute leukemia and malignant lymphoma (36 courses). In the beginning of marrow suppression, HFR and/or MFR were decreased faster than LFR, neutrophils or platelets in 14 courses (38.9%). Simultaneous decrease of HFR, MFR and other blood cells were observed in 57.2%. In the recovery phase of bone marrow, faster increase of HFR and/or MFR was noted in 52.8% and simultaneous increase of these reticulocytes with other blood cells was observed in 9 courses (25%). Appearance rate of RNA-rich younger reticulocytes is a sensitive parameter for the prediction of bone marrow suppression and recovery during the chemotherapy for hematologic malignancy.

Expression of CD90 on keratinocyte stem/progenitor cells.

BACKGROUND: The identification and purification of keratinocyte stem cells (KSCs) that are capable of self-renewal and maintenance of differentiating cell populations could contribute both to our understanding of the biology of these cells, and to significant clinical applications, such as the culturing of keratinocytes for transplantation to severe burn wounds. Here, we report the detection of CD90(+) cells in cultured normal human epidermal keratinocytes and adult skin. OBJECTIVES: To investigate the biological function of CD90(+) and CD90(-) keratinocytes. METHODS: CD90(+) and CD90(-) keratinocytes were purified from adult skin and cultured keratinocytes using fluorescent activated cell sorting, and their biological abilities were analysed using both in vitro and in vivo assays. RESULTS: Flow cytometry (FCM) analysis identified approximately 18% of post-primary neonatal keratinocytes as CD90(+). However, during expansion of the culture, the expression level of CD90 rapidly decreased to about 2.5% at passage 10, while most of the keratinocytes maintained expression of alpha6 integrin. Purified CD90(+) keratinocytes demonstrated a sixfold higher cell growth rate than CD90(-) cells and the ability to form large (over 3 mm in diameter) colonies. We then quantitatively evaluated both populations using a previously described in vivo human epidermal cyst formation assay. Enhanced green fluorescent protein (EGFP)-labelled CD90(+) or CD90(-) keratinocytes were subcutaneously injected into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Six weeks after transplantation, EGFP(+) cell clusters in human epidermal cysts were evaluated using image analysis software. EGFP(+) cell cluster areas in the basal layer, derived from EGFP(+) CD90(+) cells, were eightfold larger than clusters of EGFP(+) CD90(-) cells. Furthermore, immunohistochemical staining and FCM analysis indicated that CD90 was expressed in most of the basal layer of the normal human epidermis. CONCLUSIONS: These results indicated that CD90 is a useful marker for the detection of human KSC-enriched populations in cultured human keratinocytes.

Isolation and characterization of murine clonogenic osteoclast progenitors by cell surface phenotype analysis.

Osteoclasts are bone resorbing cells of hematopoietic origin; however, a progenitor cell population that gives rise to mature osteoclasts remains elusive. We have characterized a unique cell surface phenotype of clonogenic osteoclast progenitors (colony-forming unit-osteoclast [CFU-O]) and obtained a marrow cell population selectively enriched for these progenitors. Whole bone marrow cells were sequentially separated based on physical and cell surface characteristics, and the presence of CFU-O and other hematopoietic progenitors was examined. CFU-O was enriched in a nonadherent, low-density, lineage-marker-negative (Lin-), Thy1.2-negative (Thy1.2-), Sca1-negative (Sca1-), and c-kit-positive (c-kit+) population, as were the progenitors that were responsive to macrophage-colony-stimulating factor(CSF; CFU-M), granulocyte-macrophage-CSF (CFU-GM), and stem cell factor (CFU-SCF). When the Lin-Thy1.2-Sca1- population was divided into c-kithigh and c-kitlow populations based on c-kit fluorescence, over 88% of CFU-M, CFU-GM, and CFU-SCF were found in the c-kithigh population. In relation to the above mentioned hematopoietic progenitors, CFU-O was significantly higher in the c-kitlow population: 80% of progenitors present in the c-kitlow population were CFU-O. The CFU-O in both c-kithigh and c-kitlow populations showed key features of the osteoclast: multinucleated tartrate-resistant acid phosphatase-positive cell formation, expressions of vitronectin receptors, c-src and calcitonin receptors, and bone resorption. We have identified a progenitor cell population in the earliest stage of the osteoclast lineage so far described and developed a method to isolate it from other hematopoietic progenitors. This should help pave the way to understand the molecular mechanisms of osteoclast differentiation.

A human prostatic growth factor (hPGF): partial purification and characterization.

A growth factor capable of stimulating DNA synthesis of BALB/3T3 cells was purified about 1,000-fold from the cytosol of human benign hypertrophic prostates by heparin-Sepharose chromatography; the growth factor bound to the column in the presence of 0.5 M NaCl was eluted with 1.5-1.7 M NaCl. Its molecular weight and isoelectric point were estimated to be 11,000-13,000 and 10.5, respectively. It was sensitive to heat- and acid-treatments but resistant to disulfide-reducing agent. The final preparation was able to stimulate DNA synthesis at 10 ng/ml. The degree of stimulation was dependent on serum concentration in the assay system; the degree of maximum stimulation increased about 5 times as serum concentration increased from 0.2 to 2%.

Stabilization of fibroblast growth factors by a non-cytotoxic zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS).

The potential usefulness of a zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHPAS), in the stabilization of acidic and basic fibroblast growth factors (FGFs) was examined. Among several detergents, CHAPS was found to be not only non-cytotoxic but also most useful in handling the diluted preparations of FGFs. The advantages are as follows: 1) at lower concentrations than 0.01% CHAPS did not affect growth factor activity of calf serum (CS) and the growth rate of BALB/c 3T3 cells. The primary culture of rat prostate epithelium and colony formation of NRK-49F cells were hardly influenced by CHAPS lower than 0.003%; 2) the loss of FGFs that usually occurs due to their adherence to the surface of storage containers was effectively prevented by inclusion of 0.1% CHAPS; 3) the recovery of FGFs after storage or dialysis was significantly enhanced by inclusion of 0.1% CHAPS; 4) CHAPS at lower concentrations than 0.1% does not interfere with amino acid analysis, except that Thr may be misled only when the ratio of protein/CHAPS is low; 5) amino acid sequence analysis was hardly disturbed by CHAPS up to 0.5%. These results indicate that CHAPS is useful as a stabilizing agent for various kinds of polypeptides capable of showing biological activity at a low concentration.

Differences in nonhistone protein changes in rat ventral and dorsolateral prostate during sexual maturation.

Age-related changes of chromosomal proteins in the dorsolateral and ventral prostates of rats from 6 to 31 weeks of age were studied by SDS-polyacrylamide gel electrophoresis. A nonhistone protein having a molecular weight of about 20,000 (20K-NHP), abundantly localized in the dorsolateral prostate, increased rapidly in content during the early stage of sexual maturation (6-11 weeks of age) in association with increases of serum testosterone concentration and prostatic tissue weight. Serum testosterone concentration decreased after week 11 and then remained constant until week 31. In contrast, the 20K-NHP content continued to increase after 11 weeks of age in the dorsolateral prostate, but not in the ventral prostate. The rapid increase of 20K-NHP in the dorsolateral prostate during the early stage of sexual maturation could not be attained in immature rats (5 weeks of age) by injection of excess amounts of androgens and/or prolactin for a week. But the 20K-NHP content in the ventral prostate of rats treated with testosterone propionate was almost the same as that of mature rats.

Localization of prostatic basic protein ("probasin") in the rat prostates by use of monoclonal antibody.

Isolated nuclei of the rat prostates contain a unique androgen-dependent basic protein, "probasin". Despite that it was hardly detectable in the cytosol centrifugally prepared from the prostates, immunofluorescent histological analysis of whole tissues using monoclonal antibody, which was raised against probasin purified from the nuclei, revealed that probasin was abundantly localized in the lumen and acinal regions of the epithelium, but hardly in the nuclei. Previous extraction of secretory fluid from the prostates caused about 60% decrease in the probasin content of isolated nuclei. These suggest that probasin was originally a secretory component in the prostates, being redistributed from the secretory fluid and granule into nuclei during fractionation of subcellular components.

Conditions that support long-term production of osteoclast progenitors in vitro.

To better understand the mechanisms of osteoclast precursor development from hematopoietic stem cells, we examined the conditions that support the production of osteoclast progenitors, osteoclast colony-forming units (CFU-O), from long-term bone marrow cultures established under myeloid (Dexter's) and lymphoid (Whitlock and Witte's) conditions. Nonadherent cells harvested weekly from myeloid or lymphoid long-term cultures were assayed for CFU-O-derived colony formation in agar in the presence of a murine osteoclast colony-stimulating factor. The myeloid system supported CFU-O production for weeks, but the system produced many other types of myeloid colonies and cells as well, and quantification of CFU-O-derived colonies was difficult. The lymphoid long-term culture system also produced CFU-O; however, CFU-O production in the lymphoid system appeared more selective than in the myeloid system, but was transient. Interestingly, the addition of medium containing G-CSF to these cultures greatly enhanced (> 200%) the CFU-O production. This enhanced CFU-O production was confirmed using bone marrow cultures established on a defined marrow stromal cell line under lymphoid conditions and supplemented with recombinant murine G-CSF. Thus, G-CSF facilitates the development of clonogenic osteoclast progenitors from hematopoietic stem cells in lymphoid long-term culture conditions. This culture system may serve as a useful model for ex vivo generation of osteoclast progenitors.

The usefulness of CHAPS as a non-cytotoxic stabilizing agent in purification of growth factors.

Among several detergents, a zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), was found to be least cytotoxic for cultured mammalian cells. CHAPS improved the activity recovery and elution profile of crude and purified fibroblast growth factors (FGFs) during chromatographies. Diluted preparations of FGFs were stabilized by CHAPS against the loss during storage. Amino acid sequence analysis was not disturbed by CHAPS. CHAPS was removable by reversed-phase high-performance liquid chromatography. These results indicate that CHAPS is useful as a non-cytotoxic stabilizing agent in purification of various kinds of bioactive polypeptides.

Lobe-specific distribution of a 20,000-dalton nonhistone protein in the dorsolateral prostate of rats.

Distribution of an androgen-dependent, 20,000-dalton nonhistone protein with a pI of about 11.5 (20K-NHP) was examined by electrophoretic techniques. Nuclei of the brain, liver, spleen, skeletal muscle, lung, and thymus of rats contained a negligible amount of 20K-NHP, whereas 20K-NHP was distinctly detectable, in different relative amounts, in the nuclei of the male accessory sex organs, with the dorsolateral prostate having the highest relative content (100%), followed by the coagulating gland (approximately equal to 16%), the ventral prostate (approximately equal to 6%), and the seminal vesicle (approximately equal to 2%). There were heterogeneous distributions of cytosol components, acid phosphatase isozymes, and nonhistone proteins in the dorsolateral prostate. Zinc was localized in the lateral lobe, and fructose and glucose were in the dorsal lobe. Cytosol proteins with pI 7.5, 8.2, and 8.5 were abundant in the dorsal lobe, and proteins with pI 7.4 and 8.0 in the lateral lobe. Acid phosphatase isozymes with pI 7.1, 7.4, 7.7, and 8.0 were abundantly distributed in the lateral lobe. Of the nonhistone proteins, 20K-NHP showed the highest content both in the lateral lobe and in the dorsal lobe. It was found that 20K-NHP was more abundantly distributed in the lateral lobe (maximally four times higher) than in the dorsal lobe. The heterogeneous distribution of 20K-NHP in the dorsolateral prostate was strikingly similar to that of zinc. It appears, therefore, that 20K-NHP is closely related to dorsolateral prostate zinc content.

Enrichment of lineage-CD34- cells using a newly developed filter system.

Recent studies have demonstrated the presence of human lineage- (lin-)CD34- cells that are capable of differentiating into CD34+ cells in xenogeneic transplantation systems. We developed a new filter system that can enrich lin-CD34- cells, a precursor cell population of CD34+ cells. The filter consists of polyethylene terephthalate non-woven fabrics coated with hydrophilic polymer. The frequency of lin-CD34- cells in the cell population after filtration was 7.45 +/- 4.41%, a 16.8 +/- 8.81-fold enrichment compared with 0. 49 +/- 0.31% in the cell population before filtration. The mean recovery of lin-CD34- cells was 48.57 +/- 13.59% (n = 15). Purified lin-CD34- cells, obtained by sorting the filtrated cell population, acquired CD34 expression and colony-forming activity after 7 d of culture. Our results indicate that this filter system is useful for isolating lin-CD34- cells, including precursors of CD34+ cells, and will help further the study of lin-CD34- cells.

Changes of an androgen-dependent nuclear protein during functional differentiation and by dedifferentiation of the dorsolateral prostate of rats.

Nuclei of the dorsolateral prostate of rats contain a large amount of androgen-dependent non-histone protein (20K-NHP) (mol. wt. not equal to 20,000; pI not equal to 11.5) (Matuo et al. (1]. Its content in the nuclei increased most markedly during 4-8 weeks of age, when functional differentiation of the prostate was most active on the basis of the changes of major cytosol proteins and zinc. Nuclei of the Dunning tumors originating in the dorsolateral prostate were found to lack 20K-NHP regardless of androgen dependency, indicating the disappearance of the 20K-NHP from the nuclei by dedifferentiation. These suggest that the 20K-NHP is an important nuclear protein for differentiation of the dorsolateral prostate cells.

Comparison of subcellular proteins of normal prostate, benign prostatic hypertrophy, and prostatic cancer: presence of BPH-associated nonhistone proteins.

Proteins in the cytosol, postnuclear particulate, and nuclear fractions from seven specimens of normal prostate from bladder cancer patients, 14 specimens of benign hypertrophic prostate (BPH), and three specimens of cancerous prostate were analyzed and compared by SDS-polyacrylamide slab gel electrophoresis. Abundant protein species in the cytosol fractions were 60K (species having a molecular weight of about 60,000) and 42K; their relative contents were about 35% for 60K and about 12% for 42K. In the postnuclear particulate fraction, 42K was the most abundant (about 10% of the total). The contents of these major protein species were similar in specimens of normal and diseased prostates. In addition, there are marked similarities in the electrophoretic patterns for all the protein (24-29 species) in the cytosol and postnuclear particulate fractions of the human prostate, except for four minor species in the cytosol fraction. Of the nuclear proteins, the content of core histones (H2A, H2B, H3, and H4) was fundamentally similar among all the specimens, whereas the content of H1 histone was different from one specimen to another. The most remarkable and significant difference was that the 42K-NHP (nonhistone protein having a molecular weight of about 42,000), 55K-NHP, and 190K-NHP concentrations were significantly higher in BPH than in normal and cancerous prostates.