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Recent studies describe beneficial effects of bone marrow-derived mesenchymal stem cell infusion in animal models as well as in patients. However, data on the homing abilities of primary and culture-expanded MSC are lacking. In order to systematically investigate MSC homing we compared the fate of both primary and cultured MSC in a syngeneic mouse model. Twenty-four hours after transplantation of uncultured EGFP-transgenic MSC into sublethally irradiated mice, as many as 55-65% of injected CFU-F were recovered from the BM and 3.5-7% from the spleen. In the subsequent 4 weeks these donor CFU-F expanded 100-fold, which resulted in a normalization of femoral and splenic CFU-F numbers. This highly efficient homing of primary CFU-F contrasted with the defective homing of MSC following culture. Following their infusion immortalized multipotent syngeneic stromal cells were undetectable in BM, spleen, lymph nodes or thymus. Remarkably, following transplantation of primary MSC that had been cultured for only 24 h the seeding fraction in the BM was reduced to 10%, while after transplantation of 48 h cultured primary MSC no CFU-F were detected in the lymphohematopoietic organs. These data suggest that in vitro propagation of BM-derived MSC dramatically decreases their homing to BM and spleen.

The aim of this study was to develop an inexpensive method for reducing the number of differentiated cells from granulocyte colony-stimulating factor-mobilized leukocytapheresis products (LPs) containing peripheral blood stem cells. Analysis of LPs showed the presence of significant numbers of monocytes and myeloid cells. The myeloid cells represented largely immature stages of the granulocyte lineage (myelocytes and metamyelocytes). We investigated whether these cells could be selectively depleted by filtration over nylon wool. Filtration of LP samples over nylon wool in a medium containing fetal calf serum resulted in variable but on average low yields of CD34+ cells (48 +/- 30%; n=13) and strongly variable depletions of myeloid cells. The adherence of CD34+ cells to the polyamide fiber was partially mediated by activated platelets that were present in the LPs. Removal of platelets by counterflow centrifugal elutriation before filtration resulted in increased yields of CD34+ cells in the filtrates (65 +/- 13%; n=10). The yield of progenitor cells was similarly enhanced when trisodium citrate, a chelating substance, was added to the filtration medium. Adherence of the myeloid cells to the nylon fiber was promoted by preincubation of the columns with human immunoglobulin (Ig) (2 mg/mL). Small-scale filtrations of LP samples in the presence of trisodium citrate over columns with Ig-coated nylon wool resulted in removal of 96 +/- 4% of the monocytes and 74 +/- 18% of the myeloid cells, with a yield of 71 +/- 15% CD34+ cells and 67 +/- 10% granulocyte-monocyte colony-forming units (CFU-GM) (n=23). There was no loss of primitive stem cells during the procedure: the yield of late-appearing cobblestone area-forming cells (CAFCs, week 6) was 110 +/- 30% (n=4). CFU-GM production per CAFC-derived clone was unchanged upon filtration, indicating that the quality of stem cells was not affected. Moreover, the proportions of CD34+ cells expressing a primitive immunophenotype (CD38low or Thy-1+) were unchanged after filtration. Further enrichment of progenitor cells was obtained by separation of LP samples by elutriation before filtration. The combination of these two techniques resulted in complete removal of platelets, 89 +/- 7% depletion of erythrocytes, and 91 +/- 6% reduction of leukocytes, with a 50% yield of CD34+ cells (n=14). In conclusion, we have developed a rapid filtration technique by which monocytes and myeloid cells can be depleted from LP samples, with only minor loss of colony-forming cells and complete recovery of primitive stem cells.

Stroma-supported long-term cultures (LTC) allow estimation of stem cell quality by simultaneous enumeration of hematopoietic stem cell (HSC) frequencies in a graft using the cobblestone area forming cell (CAFC) assay, and the ability of the graft to generate progenitors in flask LTC (LTC-CFC). We have recently observed that the number and quality of mobilized peripheral blood stem cells (PBSC) was low in patients having received multiple rounds of chemotherapy. Moreover, grafts with low numbers of HSC and poor HSC quality had a high probability to cause graft failure upon their autologous infusion. Because ex vivo culture of stem cells has been suggested to present an attractive tool to improve hematological recovery or reduce graft size, we have studied the possibility that such propagation may affect stem cell quality. In order to do so, we have assessed the recovery of different stem cell subsets in CD34+ PBSC after a 7-day serum-free liquid culture using CAFC and LTC-CFC assays. A numerical expansion of stem cell subsets was observed in the presence of interleukin-3 (IL-3), stem cell factor, and IL-6, while stroma-contact, stromal soluble factors, or combined addition of FLT3-ligand and thrombopoietin improved this parameter. In contrast, ex vivo culture severely reduced the ability of the graft to produce progenitors in LTC while stromal soluble factors partly abrogated this quality loss. The best conservation of graft quality was observed when the PBSC were cultured in stroma-contact. These data suggest that ex vivo propagation of PBSC may allow numerical expansion of various stem cell subsets, however, at the expense of their quality. In addition, cytokine-driven PBSC cultures require stroma for optimal maintenance of graft quality.

Monoclonal antibody ER-MP12 defines an antigen (Ag) on murine hematopoietic stem cells that is differentially expressed by the various subsets in the hematopoietic stem cell compartment. To test whether ER-MP12 could be an asset for further subfractionation of these subsets, we physically sorted our previously defined low-density ER-MP20- (i.e., Ly-6C-) Rhodamine-123dull (Rh123dull), and wheat germ agglutinindim (WGAdim) stem cell populations on the basis of ER-MP12 Ag expression. In addition, we determined the distribution of the ER-MP12 Ag on bone marrow 6 days after 5-FU treatment. Long-term and transiently repopulating stem cell subsets were both identified in vitro using the cobblestone area-forming cell (CAFC) assay. The data show that sorting on the basis of ER-MP12 improves the separation of primitive and more mature stem cell subsets in the Rh123dull but not in the WGAdim subpopulation. However, the combination of sorting cells on the basis of an intermediate ER-MP12 expression and a low WGA affinity (ER-MP12mediumWGAdim) allows an 840-fold enrichment for in vitro long-term repopulating cells (day-28 CAFC) when compared with unseparated bone marrow. The distribution of the ER-MP12 Ag on 5-FU-treated bone marrow stem cells was similar to that in normal bone marrow stem cells, suggesting that the level of Ag expression is not dependent on cell-cycle status. Together, the combination of ER-MP12 and WGA offers the advantage of a positive selection strategy for hematopoietic stem cells, allowing different stem cell subsets to be distinguished on the basis of their primitiveness. Since no mature bone marrow cells are found within the WGAdimER-MP12medium subpopulation, the combination of ER-MP12 and WGA enables hematopoietic stem cells to be highly enriched and thus makes the use of a cocktail of lineage-specific antibodies redundant.

The cobblestone-area forming cell (CAFC) assay permits a direct measurement of the seeding of primitive and more mature murine hematopoietic stem cell subsets by comparing the number of CAFC in the original transplant with the number of CAFC retrieved from bone marrow (BM) and spleen after transplantation. We found no differences in seeding efficiency between the more mature and primitive CAFC subsets, nor between seeding efficiencies of stem cells from low-density (LD) fractions of normal and day-6 post-5-fluorouracil BM. The data show that 18% to 20% of all intravenously transplanted stem cell subsets seed to the BM, whereas 8% to 10% seed to the spleen. In addition, similar seeding efficiencies were found for day-12 spleen colony-forming unit (CFU-S-12) as was determined by retransplantation. Previously, it has been reported that a 2- to 3-hour preincubation of BM with interleukin-3 (IL-3) enhances the in vivo repopulating ability of a graft. To test whether hematopoietic growth factors affected this increased engraftment by enhancing the seeding of the transplanted marrow, we assessed the 16- to 18-hour seeding efficiency of short- and long-term in vivo repopulating stem cell subsets to BM and spleen using the CAFC assay, after preincubation with or without hematopoietic growth factors. A 2- to 3-hour preincubation with IL-3, or a combination of IL-3, IL-12, and steel factor, at 37 degrees C, led to a substantial decrease in seeding compared with control (which was kept on ice) of all hematopoietic subsets measured, both in spleen and BM. In concert with these data, the long-term in vivo repopulating ability of growth-factor incubated BM was also decreased when compared with control. In conclusion, we have been unable to observe a beneficial effect of growth factor preincubation on the repopulating ability of a graft.

Monoclonal antibody ER-MP12 defines a novel antigen on murine hematopoietic stem cells. The antigen is differentially expressed by different subsets in the hematopoietic stem cell compartment and enables a physical separation of primitive long-term repopulating stem cells from more mature multilineage progenitors. When used in two-color immunofluorescence with ER-MP20 (anti-Ly-6C), six subpopulations of bone marrow (BM) cells could be identified. These subsets were isolated using magnetic and fluorescence-activated cell sorting, phenotypically analyzed, and tested in vitro for cobblestone area-forming cells (CAFC) and colony-forming units in culture (CFU-C; M/G/E/Meg/Mast). In addition, they were tested in vivo for day-12 spleen colony-forming units (CFU-S-12), and for cells with long-term repopulating ability using a recently developed alpha-thalassemic chimeric mouse model. Cells with long-term repopulation ability (LTRA) and day-12 spleen colony-forming ability appeared to be exclusively present in the two subpopulations that expressed the ER-MP12 cell surface antigen at either an intermediate or high level, but lacked the expression of Ly-6C. The ER-MP12med20- subpopulation (comprising 30% of the BM cells, including all lymphocytes) contained 90% to 95% of the LTRA cells and immature day-28 CAFC (CAFC-28), 75% of the CFU-S-12, and very low numbers of CFU-C. In contrast, the ER-MP12hi20- population (comprising 1% to 2% of the BM cells, containing no mature cells) included 80% of the early and less primitive CAFC (CAFC-5), 25% of the CFU-S-12, and only 10% of the LTRA cells and immature CAFC-28. The ER-MP12hi cells, irrespective of the ER-MP20 antigen expression, included 80% to 90% of the CFU-C (day 4 through day 14), of which 70% were ER-MP20- and 10% to 20% ER-MP20med/hi. In addition, erythroblasts, granulocytes, lymphocytes, and monocytes could almost be fully separated on the basis of ER-MP12 and ER-MP20 antigen expression. Functionally, the presence of ER-MP12 in a long-term BM culture did not affect hematopoiesis, as was measured in the CAFC assay. Our data demonstrate that the ER-MP12 antigen is intermediately expressed on the long-term repopulating hematopoietic stem cell. Its level of expression increases on maturation towards CFU-C, to disappear from mature hematopoietic cells, except from B and T lymphocytes.

Growth and differentiation of hematopoietic stem cells occur in close contact with the cells and extracellular-matrix (ECM) proteins of the hematopoietic microenvironment. We observed the role of fibronectin, a matrix glycoprotein proposed to be involved in the attachment of hematopoietic cells and in the binding of stem cell subsets to bone marrow stroma, for this study. Murine bone marrow cells (BMC) were allowed to adhere to surfaces coated with human plasma fibronectin. Using the cobblestone-area-forming cells (CAFC) assay, adherent and nonadherent cell fractions were tested for their quantity of primitive and less primitive stem cell subsets. The CAFC assay is based on a time-dependent clone formation in pre-established, bone marrow-derived, irradiated stromal layers under limiting dilution conditions, and it allows in vitro enumeration of day-12 colony-forming unit-spleen (CFU-S12) (CAFC-10) and more primitive cells with long-term repopulating abilities (LTRA) (CAFC-28/35). We observed that the majority of primitive CAFC-28/35 adhered to fibronectin, while only a minority of CFU-S-like CAFC-10 did. The adherence of primitive stem cells to fibronectin could partially be blocked by high molar concentrations of oligopeptides containing the essential amino acid sequence of the central cell-binding domain of fibronectin, RGD. Adherence of the small subpopulation of CAFC-10 to fibronectin could almost entirely be prevented by oligopeptides organized in a specific fashion. These data suggest a role for RGD-binding integrins in the adherence of hematopoietic stem cells.

A number of different alkylating chemotherapeutic agents--busulphan, dimethylbusulphan (DMB), isopropylmethane sulphonate (IMS), melphalan, cyclophosphamide (CY) and bischloroethylnitrosourea (BCNU)--were investigated for their cytotoxic effects on different haemopoietic cell populations in host mice and for their ability to induce short- and long-term engraftment of transplanted bone marrow. At 24 h after drug treatment the femoral content of transient and permanent repopulating stem cell subsets was assessed, respectively, from the frequency of early- (day 5-15) and late- (day 25-35) developing cobblestone area-forming cells (CAFCs), growing in vitro in long-term bone marrow cultures (LTBMCs). At this time a fixed complement of 10(7) congenically marked donor bone marrow cells (B6-Gpi-1a-->B6-Gpi-1b) was infused in the drug-treated mice and erythroid engraftment was followed over 36 weeks. Diverse effects on early- and late-developing CAFC frequencies were found for the different drugs; these were generally related to the pattern of donor bone marrow engraftment in treated recipients. Melphalan was more toxic to early-developing than to late-developing CAFC subsets, and the transplant only offered an early wave of blood chimerism followed by return of host cells. CY and BCNU had minimal to moderate effects on CAFC content and engraftment with no apparent preference for any particular haemopoietic cell subset. IMS also had a relatively low toxic effect on host marrow CAFC frequencies but appeared exceptional in its ability to allow for more donor-type engraftment. The dimethane sulphonate compounds busulphan and DMB were especially potent at depleting late CAFC subsets and ensured high and lasting levels of donor bone marrow engraftment. These studies support the value of CAFC measurements for predicting the fate and growth of transplanted bone marrow cells in recipients pretreated with a variety of cytotoxic agents.

We have investigated the contribution of highly purified day-12 spleen colony-forming units (CFU-S-12) as well as more primitive cells to sustained blood cell production using in vivo and in vitro assays that allow frequency analysis. Normal or day-6 post-5-fluorouracil light-density bone marrow (BM) was sorted on the basis of differences in rhodamine-123 (Rh123) retention or wheat germ agglutinin (WGA) affinity and tested in vivo using a recently developed alpha-thalassemic chimeric mouse model. In addition, short-term and long-term clonal activity was assessed in vitro using a limiting dilution-type long-term BM culture, the cobblestone area forming cell assay. When sublethally irradiated alpha-thalassemic mice were transplanted with as many as 281 purified WGAbright CFU-S-12, derived from a fraction containing 95% of all CFU-S-12 from day-6 post-5-fluorouracil light-density BM of wild-type mice, detectable chimerism was not observed at 6 months posttransplantation. In contrast, only three CFU-S-12 were included in the Rh123dull and WGAdim subpopulations that induced 29% to 58% and 21% to 31% stable multilineage donor-type chimerism of erythrocytes and leukocytes, respectively. The Rh123dull and WGAdim cells were up to 240-fold enriched for long-term repopulating ability (LTRA) as compared with unseparated BM. A comparable level of chimerism was found in the different hematopoietic organs and at the level of BM CFU-S-12. The frequency of the LTRA unit capable of inducing a 10% sustained level of donor-type erythrocytes was calculated to be 1 to 2 per 10(5) BM cells. Several reports have suggested that LTRA and spleen colony formation could be capacities of the same stem cell subset. However, the present results show that the majority of CFU-S-12 have only short-term repopulating ability and are physically separable from more primitive stem cells with long-term multilineage reconstituting capacities.

We have studied the maintenance of stem cells with long-term multilineage repopulating ability from murine bone marrow, cultured on a pre-established bone marrow-derived stromal cell layer, both in a qualitative and quantitative way. Female bone marrow cells were cultured for a period of 1-4 weeks and compared with uncultured cells for their ability to establish and maintain a level of 50% chimerism in a sex-mismatched bone marrow transplantation model. Chimerism was determined in nucleated cells using fluorescence in situ hybridization with a murine Y-chromosome-specific probe. We observed a rapid decline in the ability of cultured marrow cells to repopulate the blood, bone marrow, spleen, and thymus of sublethally irradiated male recipients. After 4 weeks of culture only 5% of the long-term repopulating ability of the inoculated bone marrow cells remained. The remaining long-term repopulating cells, however, had similar qualities to establish and maintain long-term engraftment compared to fresh bone marrow, as judged from their ability to give stable chimerism over a period of 6 months. These observations are relevant for the therapeutic applications of long-term bone marrow cultures in purging protocols prior to autologous bone marrow transplantation of acute and chronic myeloid leukemic patients, and for the use of long-term marrow cultures when introducing foreign genetic material in hematopoietic stem cells.

In the present study we investigate the nature of the murine bone marrow cell subset responsible for the marked increase in histamine synthesis induced by interleukin-3 (IL-3). Because mast cells, and eventually their committed precursors, represent a potential source of histamine in this context, we examined their possible participation in this biologic activity with particular attention. We provide evidence that neither of these populations respond to IL-3 in terms of histamine synthesis and that other differentiated end cells or stromal components of the bone marrow are also not involved in this phenomenon. Starting from these findings, we further characterized the immature hematopoietic compartment responsible for IL-3-induced histamine synthesis using fluorescence-activated cell sorter (FACS) sorting based on rhodamine retention or wheat germ agglutinin (WGA) affinity. These procedures have allowed us to ascribe the following features to histamine-producing cells: (1) They belong to a low-density, progenitor-enriched bone marrow subset containing cells of relatively important size and internal structure. (2) The highest histamine levels are generated by the rhodamine-bright fraction of this population, while the most primitive rhodamine-dull cells do not express this biologic activity. (3) Histamine-producing cells do not copurify with colony-forming units in spleen day 7 and day 12 in WGA-bright fractions. (4) Their enrichment is associated with increased frequencies of cells forming colonies in methylcellulose (CFU-C), suggesting the involvement of several progenitors with partially limited differentiation potential in this biologic activity.

Hemopoietic stem cells show extensive heterogeneity with respect to their proliferative potential and activity. We have recently reported that the accepted technique for sorting stem cells on the basis of high affinity for the lectin wheat germ agglutinin (WGA) did not select for cells initiating long-term production of new stem cells on a stromal layer in vitro. We have therefore reinvestigated the expression of cell surface sialic acid residues in the hemopoietic stem cell compartment by sorting murine bone marrow cells on the basis of affinity for WGA. Frequency analysis of long-term bone marrow culture initiating stem cells was done using the cobblestone-area-forming cell (CAFC) assay with limiting dilution set-up. In vivo stem cell quality was determined by spleen colony formation, marrow-repopulating ability (MRA) and long-term repopulating ability (LTRA) using sex-mismatched hemopoietic chimerism. The data indicate that MRA and LTRA in vivo and in vitro are among the most WGA-dim cells. In contrast, the enrichment factors for splenic colony-forming units (CFU-S) at day 12 and transient CAFC increase with increasing WGA affinity. These characteristics allowed us to concentrate LTRA cells 590- to 850-fold over their activity in normal bone marrow without significant enrichment of day-12 CFU-S. The data reveal that WGA affinity is an inverse function of the primitiveness of murine hemopoietic stem cells and that long-term production of blood cells in vivo and in vitro is provided for by primitive cells that are physically separable from the vast majority of day-12 CFU-S. In addition the data reveal, that the CAFC frequency at day 28-35 of a graft strongly correlates with the number of cells required to induce 40% donor-type chimerism at 15 months post-transplantation and thus predicts the in vivo LTRA of a graft.

We have developed an in vitro clonal assay of murine hematopoietic precursor cells that form spleen colonies (CFU-S day 12) or produce in vitro clonable progenitors in the marrow (MRA cells) of lethally irradiated mice. The assay is essentially a long-term bone marrow culture in microtiter wells containing marrow-derived stromal "feeders" depleted for hematopoietic activity by irradiation. To test the validity of the assay as a quantitative in vitro stem cell assay, a series of unsorted and physically sorted bone marrow cells were simultaneously assayed in vivo and overlaid on the feeders in a range of concentrations, while frequencies of cells forming hematopoietic clones (cobblestone area forming cells, CAFC) were calculated by means of Poisson statistics. Linear regression analysis of the data showed high correlations between the frequency of CFU-S day 12 and CAFC day 10, and between MRA cells and CAFC day 28. A majority of MRA activity and CAFC day 28 was separable from CFU-S day 12 and CAFC day 10. This correlation study validates the CAFC system as a clonal assay facilitation both the quantitative assessment of a series of subsets in the hematopoietic stem cell hierarchy and the study of single long-term repopulating cells in vitro.

We have studied the ability of subpopulations of hemopoietic stem cells, obtained from murine bone marrow using elutriation and multiparameter sorting, to establish and maintain hemopoiesis following their deposition on irradiated stromal layers of long-term bone marrow cultures. Two fractions were obtained that differed in their mitochondrial activity as indicated by the retention of Rhodamine-123 dye. The Rhodamine-bright cell fraction, containing the majority of day-8 and day-12 spleen colony-forming units (CFU-S) and in vitro clonable progenitors, showed hemopoiesis only in the first weeks. In contrast, the Rhodamine-dull fraction, which was depleted for day-8 CFU-S and which contained the majority of cells with marrow-repopulating ability, maintained hemopoiesis for a prolonged time after an initial week of delay. These data fully support and extend previously published in vivo data, indicating that CFU-S have low capability to generate new CFU-S and granulocyte-macrophage colony-forming units (CFU-GM), and that cells responsible for long-term generation of hemopoietic precursors and for maintenance of hemopoiesis both in vivo and in vitro are the precursors of CFU-S and of in vitro clonable progenitor cells. In addition, the present findings form the basis for an in vitro assay for a primitive precursor of CFU-S, namely the marrow-repopulating cell.

Counterflow centrifugal elutriation (CCE) in combination with plastic adherence and fluorescence-activated cell sorting were used consecutively to enrich functionally different subpopulations of pluripotent hemopoietic stem cells (HSC) from mouse bone marrow. The nonadherent CCE fractions were labeled with wheat germ agglutinin (WGA)-fluorescein isothiocyanate (FITC) and sorted according to differences in fluorescence within various windows on the basis of forward (FLS) and perpendicular (PLS) light scatter. The sorted cells were then assayed for their (1) in vivo colony-forming ability (day-7 and day-12 spleen colony-forming units [CFU-S]), (2) radioprotective ability (RPA; 30-day survival), and (3) their ability to repopulate the bone marrow or spleen over a 13-day period with day-12 CFU-S, granulocyte-macrophage colony-forming units (CFU-GM), nucleated cells, or cells associated with RPA. The highest incidence of day-12 CFU-S and cells with RPA was obtained by sorting the most WGA-positive cells with relatively high PLS (enrichment, 50- to 200-fold), lowering the effective dose (ED 50/30) to an average of 80 cells. The separative procedure enabled hemopoietic stem cells that repopulate both bone marrow and spleen with secondary RPA cells, CFU-S-12, and CFU-GM to be enriched and separated from part of the RPA cells, CFU-S-12, and cells that reconstitute the cellularity of bone marrow and spleen. These data suggest that cells generating both day-12 CFU-S and RPA cells differ from day-12 CFU-S and RPA cells themselves on the basis of PLS characteristics and affinity for WGA. Such early stem cells have also been detected in sorted fractions meeting the FLS/PLS characteristics of lymphocytes.

Incubation of bone marrow cells (BMC) with neuraminidase (NA) reduced their ability to form colonies in the spleen of lethally irradiated mice to 25% of control-incubated BMC. Covering of the recipient's receptors for galactosyl residues by infusion of desialated erythrocyte membrane fragments (NA-Efr) fully prevented the reduction of NA-BMC colony formation. Upon retransplantation the desialated CFUS in the spleen of irradiated first recipients could be significantly rescued within 6 h posttransplantation by NA-Efr treatment of secondary recipients. Transplantation experiments, including NA-Efr infused and normal primary and secondary recipients, indicated that repair of the desialated CFUS cell surface probable occurred within 48 h. Establishment of growth curves indicated that desialation of CFUS did not alter their proliferative capacity but probable retarded the cells to proliferate in normal irradiated recipients for about 24 h. This might be the cause of the decreased CFUS content observed in the first recipients' spleen colonies. This delay in the onset of proliferation was not observed in mice with covered galactosyl receptors. The present experiments further indicate that (a) injected desialated CFUS are rapidly sequestrated in the recipient's body; (b) more desialated CFUS initially seed into the spleen than actually form colonies; (c) the spleen probably contains D-Galactose-specific receptors; (d) CFUS may largely restore their surface properties within 24 h following desialation, and (e) the organ distribution of normal CFUS is changed upon injection in NA-Efr infused recipients. Experiments with 51Cr-tagged BMC suggested that the organ distribution of BMC was similarly affected by neuraminidase treatment as that of CFUS.

We have studied the ability of bone marrow cell suspensions greatly differing in the relative proportion of day-7 and day-12 spleen colony-forming units (CFU-S) to rescue mice from radiation-inflicted death, and to repopulate the irradiated bone marrow and spleen with nucleated cells. Counterflow centrifugal elutriation in combination with removal of adherent cells and fluorescence-activated cell sorting on differences in wheat germ agglutinin (WGA)-fluorescein isothiocyanate (FITC) affinity and light scatter properties were used consecutively to enrich large numbers of hemopoietic stem cells from mouse bone marrow. Enrichments of 50- to 200-fold have been achieved for day-12 CFU-S and radioprotective ability (RPA), permitting 50% of lethally irradiated mice to survive over a period of 30 days with as few as 50-80 donor cells. The ratio of day-7 and day-12 CFU-S in the various suspensions could be significantly modulated on the basis of their WGA binding and perpendicular light scatter characteristics. This finding enabled us to investigate the properties of day-7 and day-12 CFU-S with respect to their RPA. We found a highly significant log/log relationship between enrichment factors for (1) RPA, (2) the number of day-12 CFU-S, and (3) spleen cellularity as measured on day 13. In addition, similar numbers of sorted and unfractionated day-12 CFU-S were required to obtain the same level of protection. Enrichment for RPA was significantly less related to either the number of day-7 CFU-S injected, or the bone marrow cellularity of the irradiated mice on day 13.

We have studied the in vivo proliferation and differential properties of murine bone marrow cells (BMC) differing in mitochondrial activity. Following centrifugal elutriation, the cells were sorted on the basis of rhodamine-123 (Rh) fluorescence intensity within a predetermined light scatter window. Unfractionated BMC colonized the spleen with a characteristic preponderance of erythrocytic nodules upon infusion into heavily irradiated mice. In contrast, Rh-dull BMC formed few macroscopic spleen colonies up to day 12, but relatively more microscopic colonies when spleens were sectioned, and the majority of the colonies showed megakaryocytic (Meg) and/or granulocytic (G) differentiation. On day 16 many megakaryocytes were found in these spleens, and very large erythroid colonies had developed since day 12. In contrast, Rh-bright BMC were highly enriched for cells forming erythrocytic spleen colonies that disintegrated after day 12, but the percentage of G and Meg colonies was low. Spleens obtained from recipients of Rh-bright cells contained a negligible number of megakaryocytes on day 16. Apparently, the mitochondrial content of cells that form hemopoietic colonies in the spleen related to the onset and duration of their lineage expression, and to the relative sizes of the lineage compartments. Thus, the colony founders of the majority of spleen surface nodules observed on days 8 and 12 are cells with high mitochondrial activity forming transient erythrocytic nodules. Such spleen colonies represent the bulk of the countable nodules that form the basis of the widely applied murine "stem cell" assay.

Mouse bone marrow cells (BMC) were subjected to countercurrent centrifugal elutriation and subsequently separated on the basis of light scatter and fluorescence intensity after being labeled with the supravital dye Rhodamine 123 (Rh-123). The sorted cells were then assayed for their in vivo spleen colony-forming ability (day -8, -12, and -16 CFU-S) and their ability to repopulate the bone marrow or spleen over a 13-day period with CFU-S-12, CFU-GM, or nucleated cells. Cells with marrow repopulating ability (MRA), as measured by the ability of the sorted cells to repopulate the marrow with secondary CFU-S-12 or CFU-GM, had low affinity for Rh-123. These cells showed minimal spleen colony-forming ability, and the ratio of MRA to CFU-S-12 in this preparation was 309. Cells with spleen repopulating ability (SRA), CFU-S-16, CFU-S-12, and CFU-S-8 retained increasing amounts of Rh-123, respectively, and CFU-S-8 were almost exclusively found among cells with high Rh-123 affinity. These cells also included about half of all day-12 CFU-S, and the ratio of MRA to day-12 CFU-S was 0. The results show that MRA cells, SRA cells, CFU-S-16, CFU-S-12, and CFU-S-8 can be sequentially ordered on the basis of increasing mitochondrial activity. The data also demonstrate for the first time, and without the application of negative selection by the use of cytostatic agents, that MRA cells are a separate class of primitive hemopoietic stem cells that fully meet the criteria of pre-CFU-S.

Incubation of bone marrow cells (BMC) with neuraminidase (NA) reduces their ability to form colonies in the spleen of lethally irradiated mice. In vivo the largest reduction was observed in the erythrocytic colonies, whereas in vitro an inhibiting effect of NA incubation was observed on the plating efficiency of erythrocyte precursors (CFUE and day 7 BFUE). Masking of the receptors for neuraminidase-treated cell surface determinants in the recipient's body by infection of desialated erythrocytes (NA-Ery) or erythrocyte membrane fragments (NA-Efr) did largely restore the reduced colony formation of NA-BMC with respect to both the total number of spleen colonies and their type. Pretreatment of irradiated host with NA-Ery, but with NA-Efr, led to a slight polycythemia as judged by the number of erythrocytic and undifferentiated colonies as well as by the surface colony diameter. The reduced erythrocytic colony formation of NA-BMC was considerably enhanced in anemic irradiated recipients (from 25-70% of respective controls). Under all experimental circumstances the erythrocytic colony formation of NA-BMC never exceeded that of normal BMC (N-BMC). Further, in normal or anemic recipients whether or not treated with NA-Efr, the diameters of the spleen surface colonies in NA-BMC injected animals were smaller than in recipients of control-incubated BMC. In vitro experimentation indicated that the reduced plating efficiency of CFUE and BFUE following incubation could not be attributed to a decreased sensitivity to erythropoietin. However, day 7 BFUE with deficient cell surface sialic acid residues had a decreased sensitivity to burst promoting activity. Among several other explanations, our data support the possibility that the desialated colony forming cells that give rise to erythrocytic colonies in vivo have higher requirements for early regulatory factors than granulocytic precursor cells. In contrast to the normal postirradiation situation the level of these factors could be increased in recipients, which are bled subsequently to irradiation. Evidence is presented in support of our concept that neuraminidase-treated CFU are fully capable to exhibit normal, although delayed, colony formation in vivo in animals with covered receptors for galactosyl residues.

Several properties of fibroblastic colony-forming units (CFU-F) from murine bone marrow and their in vitro progeny were evaluated. CFU-F had a high buoyant density relative to total bone marrow cells; they were noncycling in situ and adhered to nylon wool. The fibroblastic cells stained positively for fibronectin, lipid, alkaline phosphatase, and nonspecific esterase, while phagocytosis assays were negative, and ultrastructural analysis failed to reveal desmosomes. These properties contrasted bone-marrow-derived fibroblastic cells to both endothelial cells and macrophages. Fibroblastic cells derived from several hemopoietic organs and skin were screened for antigenic determinants present on hemopoietic cells using monoclonal antibodies. Mac-1 and B220 were absent from all fibroblastic cells studied, whereas the Forsmann and Pgp-1 antigens were always present. Thy-1 was not detected on bone-marrow-derived fibroblasts, but was present on fibroblastic cells derived from other sources. T200 was found on all hemopoietic organ-derived fibroblastic cells, but not on those derived from blood and skin. Thus, analysis of antigenic determinants allowed distinction between fibroblastic cells from different organs.