A stromal cell line from myeloid long-term bone marrow cultures can support myelopoiesis and B lymphopoiesis

The production of B lymphocytes and myeloid cells occurs in the bone marrow in association with a supporting population of stromal cells. To determine whether these processes are dependent upon the same or different populations of stromal cells, stromal cell lines were generated from the adherent layer of a Dexter type long-term bone marrow culture. These cultures support myeloid cells and their precursors, a B cell precursor, and the adherent layer cells with support B cell differentiation under appropriate conditions. Two of the lines examined, S10 and S17, express class I histocompatibility antigens but not other hemopoietic cell surface determinants such as Thy-1, Lyt-1, Ig, Ia, Mac-1, or BP-1. Both lines could support myelopoiesis under Dexter conditions upon seeding with nylon wool-passed bone marrow. The nylon wool passage depletes stromal cells capable of forming adherent layers in vitro but retains hemopoietic precursors. The number of cells and colony-forming units-granulocytes/macrophages in the nonadherent cell population recovered 3 wk post-seeding had increased 19-fold and 10-fold, respectively, in the reseeded cultures of S10 and S17. After 3 wk of growth in Dexter conditions, the reseeded cultures were transferred to conditions optimal for B cell differentiation described by Whitlock and Witte. After 4 wk of growth, hemopoietic cells were consistently recovered from S17 cultures but not those of S10. A proportion of these cells from S17 cultures expressed the 14.8 antigen and were surface IgM positive. Surviving hemopoietic cells present in cultures of S10 were primarily macrophages. These findings indicate that S17 but not S10 can support both myelopoiesis and B lymphopoiesis and suggest that one stromal cell population has the capacity to form a hemopoietic microenvironment for both lineages.     

Blast colony-forming cell binding from CML bone marrow, or blood, on stromal layers pretreated with G-CSF or SCF

Blast colony-forming cells (CFU-BL) represent a specific subpopulation of special primitive progenitors characterized by colony formation only in close contact with a preformed stromal layer. CFU-BL derived from bone marrow of chronic myeloid leukaemia (CML) patients have been proved to adhere poorly to bone marrow derived stromal layers suggesting that the appearance of progenitors and precursors in the circulation is due to a defective adhesion of these cells to the bone marrow microenvironment. In the present experiments the effect of short-term incubation of preformed normal bone marrow stroma on the adherence of CML derived CFU-BL was studied. For stroma cultures bone marrow cells were cultured in microplates in the presence of hydrocortisone. Cultures were used when stromal layers became confluent and no sign of haemopoiesis could be observed. CFU-BL were studied by panning plastic non-adherent mononuclear (PNAMNC) bone marrow or blood cells. 8.9 +/- 2.4 colonies/103 PNAMNC (six experiments) were formed from normal bone marrow on stromal layers and 4.8 +/- 2.1 colonies/103 PNAMNC (five experiments) from CML bone marrow. Colony formation from normal bone marrow was not increased if stromal layers were incubated with 100 ng/mL granulocyte colony-stimulating factor (G-CSF) or stem cell factor (SCF). Incubation of stroma with G-CSF or SCF, however, increased the colony formation of PNAMNC from CML bone marrow or blood significantly. These findings suggest that local concentration of haemopoietic growth factors at the time of panning may influence the attachment of CML progenitors to the stroma.     

Effect of opioid peptides on proliferation and differentiation of skeletogenic cells of rabbit bone marrow in vitro

The influence of opioid peptides DSLET and DAGO in doses 10(-5), 10(-7) or 10(-10) mg per 1 ml of the medium on colony formation in the culture of stromal bone marrow fibroblast precursors was investigated 5. 10(-6) bone marrow cells were placed in plastic containers (Costar). 12 day old cell cultures were fixed with ethanol and stained with hematoxyline-eosin. Effectiveness of fibroblast colony formation (EFFC) was detected. Grown fibroblast colonies were stained after Gomory for alkaline phosphatase. Opioid peptides DSLET and DAGO in the used doses exerted no influence on EFFC and percentage phosphatase-positive colonies, which casts doubt on a presumable direct action of opioid peptides on stromal bone marrow cell-precursors. But it does not seem unlikely that opioid peptides may affect stromal bone marrow precursors of fibroblasts through the cell environment, particularly, via macrophages.     

Bone marrow stromal cell lines with lymphopoietic activity express high levels of a pre-B neoplasia-associated molecule

Bone marrow stromal cell lines have been isolated that directly support B lymphopoiesis in vitro. Single B-lineage precursors proliferate and differentiate on certain of these stromal cell lines to establish long-term B-lineage cultures. These lymphopoietic stromal cells produce novel soluble factors that support proliferation of in vitro established pre-B cell populations. Lymphoid populations established on lymphopoietic stromal cell lines lack surface Ig-bearing cells, but give rise to surface Ig+ cells when transferred to mixed bone marrow feeder layers. Several stromal lines expressed a B-lineage neoplasia marker detected by the monoclonal antibody MAb6C3. Remarkably, only the 6C3Aghi stromal lines supported long-term proliferation of B-lineage cells. We propose that the 6C3 antigen-bearing molecule may play a role in stromal cell-dependent, pre-B cell proliferation, as well as in neoplastic proliferation of pre-B leukemias.     

The age-dependent loss of bone marrow B cell precursors in autoimmune NZ mice results from decreased mitotic activity, but not from inherent stromal cell defects

The formation of B lymphocytes is abnormal in autoimmune NZB and (NZB x NZW)F1 mice. With age, the proportion of sIg- Ly-5(220)+ pre-B cells and less mature B cell progenitors in the bone marrow progressively declines, reaching only approximately one-third of normal levels in 20-wk-old NZ mice. To determine the mechanisms responsible for the deficiency of NZ B lineage precursors, the mitotic activity of sIg- Ly-5(220)+ bone marrow cells in vivo was determined in NZ and conventional inbred mice as a function of age. The proportion of sIg- Ly-5(220)+ B cell precursors in (S + G2/M) stages of the cell cycle steadily decreased with age in NZ autoimmune mice. Furthermore, upon metaphase arrest, the rate of entry of sIg- Ly-5(220)+ bone marrow cells into G2/M also decreased with age in NZ mice. Therefore, the mitotic activity of sIg- Ly-5(220)+ B cell precursors is substantially decreased in NZ mice greater than or equal to 20 wk of age. The capacity of the bone marrow stromal microenvironment of NZ mice to support B lineage precursor growth was tested in two ways: 1) the capacity of preformed NZ bone marrow stroma to support B lineage cell growth in long term bone marrow cell culture under lymphopoietic conditions was assessed and 2) the capacity of NZ bone marrow B lineage precursors to expand in vivo after sublethal (200 rad) whole body irradiation was determined. Stroma derived from adult NZ mice supported the growth and development of B lineage lymphocytes in long term bone marrow cell culture to a greater extent than did age-matched conventional murine stroma. Furthermore, sublethal irradiation of older adult NZ mice resulted in some expansion of bone marrow sIg- Ly-5(220)+ B cell precursors in vivo. Therefore, the deficiency of B cell progenitors in the bone marrow of older NZ autoimmune mice is associated with diminished mitotic activity. However, this does not result from defects in the capacity of NZ bone marrow stroma to permit B lineage cell expansion as determined by both in vitro and in vivo experiments. In the absence of a detectable stromal cell defect, it is possible that an active inhibitory process within the bone marrow influences the mitotic activity of B cell precursors in NZ mice.     

Murine B-cell stimulatory factor-1 (BSF-1)/interleukin-4 (IL-4) is a multilineage colony-stimulating factor that acts directly on primitive hemopoietic progenitors

Interleukin-4 (IL-4), which was originally identified as a B-cell growth factor, has been shown to produce diverse effects on hemopoietic progenitors. The present study investigated the effects of purified recombinant murine IL-4 on early hemopoetic progenitors in methylcellulose culture. IL-4 supported the formation of blast cell colonies and small granulocyte/macrophage (GM) colonies in cultures of marrow and spleen cells of normal mice as well as spleen cells of mice treated with 150 mg/kg 5-fluorouracil (5-FU) 4 days earlier. When the blast cell colonies were individually picked and replated in cultures containing WEHI-3 conditioned medium and erythropoietin (Ep), a variety of colonies were seen, including mixed erythroid colonies, indicating the multipotent nature of the blast cell colonies supported by IL-4. To test whether or not IL-4 affects multipotent progenitors directly, we replated pooled blast cells in cultures under varying conditions. In the presence of Ep, both IL-3 and IL-4 supported a similar number of granulocyte/erythrocyte/macrophage/megakaryocyte (GEMM) colonies. However, the number of GM colonies supported by IL-4 was significantly smaller than that supported by IL-3. When colony-supporting abilities of IL-4 and IL-3 were compared using day-4 post-5-FU spleen and day-2 post-5-FU marrow cells, IL-4 supported the formation of fewer blast cell colonies than did IL-3. IL-4 and IL-6 revealed synergy in support of colony formation from day 2 post-5-FU marrow cells. These results indicate that murine IL-4 is another direct-acting multilineage colony-stimulating factor (multi-CSF), similar to IL-3, that acts on primitive hemopoietic progenitors.     

Structure and function of bone marrow environment

Bone marrow and spleen are the major hematopoietic tissue in adult mice. However, little is known about the specific mechanism regulating hematopoiesis within these tissues. Since Dexter et al. first described conditions to maintain bone marrow hematopoiesis, long term bone marrow culture (LTBMC) has been developed in order to analyze the mechanism of the maintenance of proliferation and differentiation of hematopoietic stem cells in vitro. Furthermore, several stromal cell lines which are able to support the growth and differentiation of hematopoietic lineage, has been established from LTBMC. Although it is well known that bone marrow stromal cell lines are able to produce colony stimulating factors, it has been suggested that the stromal cell factors which involve membrane bound moieties must have a key role in the regulation of hematopoiesis. We expect that monoclonal antibodies to the surface of bone marrow stromal cells could detect such a critical stroma-associated protein that bounds the cell surface of the bone marrow stroma.     

Genetic approach and phenotype-based complementation screening for identification of stroma cell-derived proteins involved in cell proliferation.

The functional capacities of stromal cell lines to support stem cell activity are heterogeneous and the mechanism of how they support bone marrow cultures remains unclear. Recently, we reported a strategy of functional analysis in which a genetic approach is combined with phenotype-based complementation screening to search for a novel secreted growth factor from mouse bone marrow stroma called ShIF that supported proliferation of bone marrow cells. To investigate the role of stromal cells in hemopoiesis, we extended this strategy to search for stroma-derived proteins that induce cell proliferation by establishing stroma-dependent Ba/F3 mutants of three stroma cell lines from two mouse tissues. Seven stroma-dependent Ba/F3 mutants were used as responder cells to identify cDNAs from stroma cell lines whose products supported proliferation not only to the mutant cells but also to hemopoietic progenitor cells in vitro.     

Pre-CFU-f: young-type stromal stem cells in murine bone marrow following administration of DNA inhibitors

Occurrence of young-type stromal stem cells (defined here as "pre-CFU-f") in murine bone marrow is reported in this study. Two consecutive intraperitoneal (i.p.) cytosine arabinoside (ara-C) injections were administered to C57B1 mice (2 X 200 mg/kg at 6-h intervals). Two days later the bone marrow was collected and assayed for colony-forming units-fibroblastoid (defined here as "CFU-f"). In additional experiments, ara-C-treated marrow was exposed in vitro to hydroxyurea (HU; "hydroxyurea killing test"), prior to plating, to establish the cycling state of stromal stem cells. In separate cultures of ara-C-treated marrow, replating of adherent cells was carried out up to quaternary sub-cultures. The results indicate ara-C-treated marrow produces approximately 20% "huge" fibroblastoid colonies (approximately 5 mm diameter versus 0.5-2 mm normal size); most stromal stem cells producing huge colonies are cycling cells; and adherent cells from primary ara-C-treated marrow cultures replated to secondary cultures produce adherent layers with double the number of cells than in the control secondary cultures. We conclude that the ara-C-treated murine bone marrow contains certain young-type cycling stromal stem cells which we refer to as pre-CFU-f. These stem cells produce huge fibroblastoid colonies in culture, indicating that they probably go through more cell cycles than CFU-f during the culture period. Alternatively, pre-CFU-f may have a higher self-replicative capacity than CFU-f.     

Normal B cell precursors responsive to recombinant murine IL-7 and inhibition of IL-7 activity by transforming growth factor-beta

The ability of stromal cells in bone marrow to support B lymphopoiesis may be partially mediated by secretion of biologically active factors. The first cytokine with lymphopoietic activity to be molecularly cloned from stromal cells, IL-7, was originally identified by its growth-promoting activity on long term cultured lymphocytes. We now report that murine rIL-7 is a potent proliferative stimulus for B cell progenitors isolated from fresh bone marrow. Proliferation was initially most obvious among large precursor cells which bear the B lineage associated Ag, Ly5/220 and BP1. A majority of these also contained cytoplasmic Ig mu H chains. Extended culture with IL-7 resulted in a predominance of immature c mu- lymphocytes. No effect by IL-7 was observed on the proliferation of mature lymphocytes. It also did not induce maturation in a number of early B lineage cell lines, or promote the formation of LPS-responsive, clonable B cells from precursors. When incorporated into semisolid agar medium, IL-7 specifically and rapidly induced the formation of pre-B cell colonies in a linear fashion with respect to numbers of cells cultured from either purified B cell progenitor preparations or unfractionated bone marrow. In both liquid and agar culture conditions, the IL-7 proliferative activity was inhibitable by two related forms of transforming growth factor (TGF) beta, TGF-beta 1 and TGF-beta 2. Taken together, these results indicate that IL-7 is a stimulus for replication of normal B lineage cells at an early stage of differentiation, and its activity can be modulated by other cytokines. IL-7 also provides a means of studying the progeny of a single B cell progenitor, and of enumerating clonable pre-B cells in the absence of colony formation by other cell types in bone marrow.     

Human marrow stromal precursors are alpha 1 integrin subunit-positive

In this work we studied the expression of adhesion molecules on primate human and non-human marrow stromal cells (primary cultures and lines) and on human CD34(+) hematopoietic normal and leukemic precursors. Differential expression of alpha1 integrin subunit was observed, since this molecule was intensely expressed by marrow stroma but not detected on CD34(+) cells. We used this difference to select, in fresh bone marrow samples, alpha 1-positive cells. We found that all stromal precursors giving rise to colony-forming units-fibroblasts (CFU-F) were present in the alpha 1-positive fraction. No colonies were detected in the alpha 1-negative fraction even after 2 weeks of culture. Phenotypic studies of stromal cells derived from alpha1-positive cells and grown in long-term marrow culture indicated that these cells were similar to stromal cells from primary cultures. We also observed early upregulation of alpha 4 and alpha 2 integrin subunits in cultures derived from alpha1-positive cells with maximal expression by day 10 (26 and 51%, respectively) preceding a gradual decline to low to nil values at day 30 (4.5 and 12%). These data indicate that alpha 1 integrin subunit is a marker for both mature stromal cells and stromal precursors, while alpha 2 and alpha 4 integrin subunits are expressed primarily by immature cells.     

Characterization of monolayer and organ cultures of cloned and enriched lymphohematopoietic stromal cell populations

The role of hematopoietic microenvironments in the regulation of maturation and differentiation of hematopoietic cells, although heavily debated, remains uncertain. Several investigators have suggested that the adherent “stromal” cell populations, which grow as colonies in cultures of lymphomyeloid tissues, include the cells involved in such regulatory processes. Grossly, the colonies described by several investigators appear similar morphologically, and the cells giving rise to them have been variously termed (1) fibroblast colony forming cells (FCFC), (2) plaque forming units-culture (PFU-C), (3) macrophage colonies, and (4) marrow stromal cells. FCFC have been reported to re-establish their parent microenvironment when transplanted in an allogeneic system. In this study, cloned and enriched cell populations obtained from such colonies in cultures of murine lymphomyeloid tissues have been characterized by their growth in culture and using morphological, histochemical, and electron microscopic techniques. The results demonstrated that, although the initial stromal colonies appeared to be identical, the constituent cell types varied considerably. Some colonies were comprised primarily of macrophages, while others appeared to contain predominantly fibroblasts; two additional cell types that established colonies have not yet been satisfactorily identified. These results demonstrate the heterogeneity of lymphomyeloid stromal colonies. There is a need for caution in the analysis of experiments in which uncharacterized stromal cell colonies are transplanted or employed as supporting monolayers in culture systems in experiments designed to evaluate the origins and functions of lymphohematopoietic stroma.     

Pre-B cell generation potentiated by soluble factors from a bone marrow stromal cell line

Two bone marrow stromal cell lines isolated from the adherent layer of a Dexter-type long term bone marrow culture differ markedly in their hemopoietic support capacity. S17 supports myelopoiesis and the differentiation of early B cell precursors into B lymphocytes while S10 supports myeloid cell differentiation and not B lymphopoiesis. The identification of a stromal cell line with B cell support capacity prompted an investigation of whether the effects of S17 were mediated via soluble factors. Results presented herein indicate that medium conditioned by S17 but not S10 contains an activity that can induce the expression of the 220,000 m.w. 14.8 antigen and cytoplasmic mu H chain of Ig in B lymphocyte progenitors that have not yet expressed these markers. Bone marrow cells were depleted of 14.8+, cytoplasmic mu+ pre-B cells on antibody-coated petri dishes. After 24-h liquid culture newly generated pre-B cells were enumerated as cells that expressed cytoplasmic mu H chain of Ig but not Ig L chains by immunofluorescence. Expression of Ly5(220) was monitored by 14.8 antibody binding. This pre-B cell differentiation activity was abrogated by digestion with pronase, aminopeptidase, or carboxypeptidase. Isoelectric focusing data revealed the activity to have isoelectric point of 5.9 to 6.2. S17-conditioned medium was fractionated using HPLC and each fraction tested for pre-B cell-generating activity. Fractions collected from a Superose 12 gel filtration column were found to have two peaks of activity associated with molecules of apparent m.w. of approximately 60,000 and 10,000. Virtually identical peaks of activity were observed when medium conditioned by heterogeneous stromal cell cultures was fractionated. Separation of S10-conditioned medium revealed no cryptic activity. S17-conditioned medium was further characterized by anion exchange chromatography and the majority of the pre-B cell generating activity shown to be associated with the void volume that eluted from a MonoQ column. These fractions were rechromatographed on Superose and the activity again found to be associated with two fractions corresponding to apparent m.w. of 60,000 and 10,000. The S17 pre-B cell differentiation activity appears to result from the presence of a novel molecule because other well characterized mediators had no activity in this short-term liquid culture system. No pre-B cell-generating activity was observed when IL-1 or conditioned medium containing IL-2, IL-3, or IL-4 (B cell stimulatory factor 1) were added to cultures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characteristics and differential T-cell dependency of human B-cell colony precursors

Studies were performed to characterize the human peripheral blood non-T cells forming colonies in semisolid cultures stimulated with Staph protein A (SpA). Negative selection experiments revealed that colony precursors largely consisted of cells bearing Fc receptors, complement receptors (CR), surface immunoglobulin (sIg), and Ia-like antigens. Most colony precursors expressed sIgM and sIgD, but not sIgG. Also, colony-forming cells were shown to be distinct from non-T cells proliferating in SpA-stimulated liquid cultures as evidenced by the greater sensitivity of colony precursors to anti-K,λ, or -Ia plus complement depletion. Two distinct categories of colony-forming cells could be distinguished by the expression of CR. CR-positive cells were responsible for greater than 85% of the colonies formed in the absence of optimal T cell numbers. Although under identical conditions CR^? cells demonstrated minimal colony growth, the addition of optimal T cell numbers significantly augmented colony responses. Thus, colony precursors express surface markers characteristic of B cells relatively advanced in the developmental pathway. However, less advanced cells are capable of colony growth in the presence of optimal T cell numbers.     

In vitro immune response of human peripheral lymphocytes. VI. Distribution and characterization of precursors for PHA- and protein A-induced colony-forming B cells

B cells from peripheral blood or cord blood formed colonies by stimulation with either PHA or protein A. On the other hand, tonsillar B cells did not form protein A-induced colonies, although PHA-induced colony formation was comparable to that observed in peripheral B cells. Lack of protein A-induced colony formation in tonsillar B cells was not due to the defect of helper T cells in preculture or to the presence of suppressor cells but was due to the absence of precursors for colony formation. The result showed that PHA- and protein A-induced colony-forming cells belonged to distinct subsets of B cells. Depletion of mu-bearing cells from peripheral B cells abrogated both PHA- and protein A-induced colony formation. Depletion of delta-bearing cells did not affect PHA- and protein A-induced colony formation and the population enriched with delta-bearing cells also showed colony formation. Depletion of complement receptor (CR)-positive cells removed precursors for both PHA- and protein A-induced colony formation. These results showed that precursor cells for PHA- and protein A-induced colony formation were IgM+, IgD+ and CR+ or IgM+, IgD- and CR+.     

Systematic analysis of the ability of stromal cell lines derived from different murine adult tissues to support maintenance of hematopoietic stem cells in vitro.

Hematopoietic stem cells interact with a complex microenvironment both in vivo and in vitro. In association with this microenvironment, murine stem cells are maintained in vitro for several months. Fibroblast-like stromal cells appear to be important components of the microenvironment, since several laboratories have demonstrated that cloned stromal cell lines support hematopoiesis in vitro. The importance of the tissue of origin of such cell lines remains unknown, since systematic generation of stromal cell lines from adult tissues has never been accomplished. In addition, the capacity of stromal cell lines to support reconstituting stem cell has not been examined. We have previously described an efficient and rapid method for the immortalization of primary bone marrow stromal cell lines (Williams et al., Mol. Cell. Biol. 8:3864-3871, 1988) which can be used to systematically derive cell lines from multiple tissues of the adult mouse. Here we report the immortalization of primary murine lung, kidney, skin, and bone marrow stromal cells using a recombinant retrovirus vector (U19-5) containing the simian virus large T antigen (SV40 LT) and the neophosphotransferase gene. The interaction of these stromal cells with factor-dependent cells Patterson-Mix (FDCP-Mix), colony forming units-spleen (CFU-S), and reconstituting hematopoietic stem cells was studied in order to analyze the ability of such lines to support multipotent stem cells in vitro. These studies revealed that stromal cell lines from these diverse tissues were morphologically and phenotypically similar and that they quantitatively bound CFU-S and FDCP-Mix cells equally well. However, only those cell lines derived from bone marrow-supported maintenance of day 12 CFU-S in vitro. One lung-derived stromal cell line, ULU-3, supported the survival of day 8 CFU-S, but not the more primitive CFU-S12. A bone marrow-derived stromal cell line, U2, supported the survival of long-term reconstituting stem cells for up to 3 weeks in vitro as assayed by reconstitution 1 year post-transplant. These studies suggest that adherence of HSC to stromal cells is necessary but not sufficient for maintenance of these stem cell populations and that bone marrow provides specific signals relating to hematopoietic stem cell survival and proliferation.     

CFU-F circulating in cord blood

CFU-F (colony forming units-fibroblast) were studied from cord blood and, as controls, from normal bone marrow of older children and adults. Numbers of CFU-F in cord blood buffy coat cells are lower by a factor of 10 in comparison to bone marrow CFU-F. Cytomorphology and staining with monoclonal antibody identify the progeny cells of CFU-F as fibroblasts. Cord blood CFU-F derived fibroblasts have properties supporting hematopoiesis: They produce CSF (colony stimulating factor) to which fresh cord blood CFU-GM (colony forming units-granulocytic, monocytic) react by colony formation in a dose-response manner. In addition, fibroblast colonies discharge clonogenic round cells into the medium forming CFU-GM and CFU-F colonies in secondary methyl cellulose cultures. We conclude that fetal blood contains clonogenic stromal cells (CFU-F) that give rise to fibroblasts with properties of hematopoietic support.     

Presence of cells in B-cell lineage in mixed (GEMM) colonies from murine marrow cells

Recent progress in clonal cell culture techniques makes it possible to detect pluripotent hemopoietic precursors from murine marrow cells. The precursors can proliferate, differentiate and form mixed colonies containing erythroblasts, granulocytes, macrophages and often megakaryocytes in viscid culture medium. In the present investigation, the presence of cells of B-cell lineage in mixed colonies was investigated. Experiments on colonies containing cIgM, cIgG, sIgM and sIgG bearing cells using goat IgG fluorescein-conjugated anti-mouse IgM, goat F(ab')2 fraction fluorescein-conjugated anti-mouse IgG and immunobeads revealed the presence of cytoplasmic IgM bearing cells in 47% of the colonies and surface IgM bearing cells in 74-84% of the colonies. Mixed colonies, however, did not contain either cIgG bearing cells or sIgG bearing cells. The results may indicate that some CFU-MIX proliferate and differentiate along B-cell lineage to sIgM or cIgM bearing cells in vitro.