Stromal support augments extended long-term ex vivo expansion of hemopoietic progenitor cells.

Current technology to numerically expand hemopoietic stem/progenitor cells (HSPC) ex vivo within 1 to 2 weeks is insufficient to warrant significant gain in reconstitution time following their transplantation. In order to more stringently test the parameters affecting HSPC expansion, we followed ex vivo cultures of CD34+-selected umbilical cord blood (UCB) HSPC for up to 10 weeks and investigated the effects of stromal support and cytokine addition. The cytokine combinations included FL + TPO, FL + TPO plus SCF and/or IL6, or SCF + IL6. To identify the HSPC in uncultured and cultured material, we determined the number of colony-forming cells (CFC), cobblestone area forming cells (CAFC), the NOD/SCID repopulating ability (SRA), and CD34+ subsets by phenotyping. The highest fold-increase obtained for CD34+ and CD34+ CD38- cell numbers was, respectively, 1197 and 30,937 for stroma-free and 4066 and 117,235 for stroma-supported cultures. In general, CFC generation increased weekly in FL + TPO containing groups up to week 5 with a 28- to 195-fold expansion whereafter the weekly CFC output stabilized. Stroma support enhanced the expansion of CAFC week 6 maximally 11-fold to 89-fold with FL + TPO + IL6. Cultures stimulated with at least FL + TPO gave an estimated 10- to 14-fold expansion of the ability of CD34+ UCB cells to multilineage engraft the BM of sublethally irradiated NOD/SCID mice at 2 weeks of stroma-free and stroma-supported cultures, while at week 5 and later the estimated SRA decreased to low or undetectable levels in all groups. Our results show that stroma and FL + TPO but also inclusion of bovine serum albumin, greatly increase the long-term generation of HSPC as measured by in vitro assays and is indispensable for long-term expansion of CD34+ CD38- CXCR4+ cells. However, the different surrogate methods to quantify the HSPC (CD34+ CD38-, CFC, CAFC week 6 and SRA) show increasing incongruency with increasing culture time, while especially the phenotypic analysis and the CFC generation greatly overestimate the CAFC and SRA expansion in 10-week cultures.

Successful short-term ex vivo expansion of NOD/SCID repopulating ability and CAFC week 6 from umbilical cord blood.

In view of the limited potential for rapid hematological recovery after transplantation of umbilical cord blood cells (UCB) in adults, we have attempted to expand CD34+ selected hemopoietic stem cells (HSC) and progenitors in 2-week cultures of whole graft pools in the presence or absence of serum and stromal layers, and with various cytokine combinations including (1) FL + TPO; (2) FL + TPO plus SCF and/or IL6; or (3) SCF + IL6. Both in the input material and cultured grafts we determined the number of colony-forming cells (CFC), cobblestone area forming cells (CAFC), the NOD/SCID repopulating ability (SRA), and CD34+ CD38- subset by phenotyping. The highest fold-increase obtained for the number of nucleated cells (nc), CD34+, CD34+ CD38 cell numbers and CFC content was, respectively, 102 +/- 76, 24 +/- 19, 190 +/- 202 and 53 +/- 37 for stroma-free and 315 +/- 110, 25 +/- 3, 346 +/- 410 and 53 +/- 43 for stroma-supported cultures. CAFC week type 6 was maximally 11-fold expanded both under stroma-free and stroma-supported conditions. The FBMD-1 stromal cells supported a modest expansion of CD34+ CD38- cells (27 +/- 18-fold) and nc (6 +/- 4-fold), while a loss of CFC and CAFC subsets was observed. The stromal cells synergized with FL + TPO to give the highest expansion of hemopoietic progenitors. Stromal support could be fully replaced by complementing the FL + TPO stimulated cultures with SCF + IL6. FL + TPO were required and sufficient to give a 10- to 20-fold expansion of the ability of CD34+ UCB cells in 2-week cultures to engraft the BM of NOD/SCID mice. Stromal support, or complementation of the medium with SCF + IL6, did not significantly improve the in vivo engraftment potential. If the SRA and CAFC week 6 assays are accepted as tentative estimates of in vivo engrafting stem cells in humans, our findings may assist in the preparation of UCB grafts to meet the requirements for improved repopulation in the clinical setting.

Interleukin-12 enhances interleukin-3 dependent multilineage hematopoietic colony formation stimulated by interleukin-11 or steel factor.

Interleukin 12 (IL-12: natural killer cell stimulatory factor, NKSF; cytotoxic lymphocyte maturation factor, CLMF) was studied for its effect on colony formation and lineage expression of low-density bone marrow cells from 5-fluorouracil-treated mice, and of sorted stem cells using a semi-solid culture assay in the absence or presence of IL-3, IL-11, Steel factor (SF) and erythropoietin. IL-12 did not support colony formation as a single factor, nor in the presence of IL-11 or SF. In IL-3-containing cultures, IL-12 slightly enhanced neutrophilic and monocyte differentiation. Both SF and IL-11 synergized with IL-3 to increase the percentage of multilineage colonies and the number of colonies containing erythrocytes, megakaryocytes, neutrophils, eosinophils, monocytes/macrophages, and blast cells, but not mast cells. In the presence of IL-3 + IL-11, IL-12 greatly enhanced neutrophil, megakaryocyte, erythrocyte, and mast cell development. In IL-3 + SF-containing cultures, IL-12 further increased colony numbers and a higher percentage of colonies expressed neutrophilic, megakaryocytic, erythroid, monocytic, blast cell, and/or mast cell lineages. Colony size and the presence of eosinophils in colonies were unaffected by IL-12 addition. These effects of IL-12 could not be reversed by antibodies against interferon-gamma. Our data show that IL-12 may act as a synergistic factor, stimulating multilineage expression of hemopoietic stem cells, probably via a direct action. The observed activity of IL-12, however, required the presence of a least two factors, i.e. either IL-3 + IL-11, or IL-3 + SF.

Interleukin-12 synergizes with interleukin-3 and steel factor to enhance recovery of murine hemopoietic stem cells in liquid culture.

Interleukin 12 (IL-12; natural killer cell stimulatory factor, NKSF; cytotoxic lymphocyte maturation factor, CLMF) was studied for its effects on the survival and generation of hemopoietic progenitors (CFU-C, CAFC day 7-14) and long-term culture initiating stem cells (CAFC day 28-35) by post-5-fluorouracil (FU) bone marrow cells (BM) in low-serum liquid culture. Previous data have shown that IL-12 may act as a potent synergistic factor stimulating multilineage expression of hemopoietic stem cells in the presence of IL-3 in combination with Steel factor (SF) or IL-11. IL-12 or IL-11 alone did not support CFU-C production in liquid culture of a low-density fraction of day-6 post-FU BM. IL-11 and SF showed potent synergy with IL-3 to augment output CFU-C numbers and support CAFC-28/35 maintenance; SF as a single factor, or in combination with IL-11 or IL-12, allowed low recovery of CFU-C and all CAFC subsets. IL-12 synergized with IL-3, and with IL-3 + SF, to moderately increase progenitor production in liquid cultures. Similarly, survival and generation of the primitive CAFC-28/35 was consistently increased when IL-12 was used in combination with IL-3, or IL-3 + SF. The combination of 3, SF and 12 supported a significantly higher recovery of CAFC-35 than did the combination of 3, SF and 11, and CAFC-35 were numerically expanded during culture. Furthermore, IL-12 acted synergistically with IL-3 to enhance CFU-C output over a prolonged period using progenitor-depleted 3-day post-FU BM. These data show that IL-12 interacts with IL-3 and SF to regulate survival and growth of myeloid stem cells and progenitor cells.

Autocrine transforming growth factor beta 1 blocks colony formation and progenitor cell generation by hemopoietic stem cells stimulated with steel factor.

The ability of Steel Factor (SF) to stimulate colony formation and progenitor cell generation by hemopoietic stem cells (HSCs) in vitro in the absence of interleukin 3 (IL-3) was investigated. IL-3 was required for HSC proliferation, and no or restricted proliferation occurred in the presence of SF, IL-6, IL-11, or IL-12 as single factors or in combination. Neutralizing concentrations of anti-transforming growth factor (TGF)-beta 1 antibodies enhanced progenitor cell generation 2-3-fold in the presence of IL-3, but 75 to over 300-fold when cultures contained at least SF in the absence of IL-3. Exogenous TGF-beta 1 fully abrogated the antibody effects. In the presence of antibodies to TGF-beta 1, SF alone stimulated the delayed formation of small blast cell colonies and SF synergized with IL-6, IL-11, or IL-12 to greatly hasten colony formation, enhance colony number and size, and increase colony forming unit-culture (CFU-C) output from suspension cultures of enriched HSC populations. Secondary CFU-C colonies were significantly larger when IL-3 was absent during the suspension culture phase. Single cell and limiting dilution analysis using a homogenous colony forming unit-spleen (CFU-S) day-12 population and an 800-fold enriched long-term repopulating HSC fraction, respectively, indicated that TGF-beta 1 was an autocrine product of these HSC subsets. Addition of nucleosides, insulin, extra glucose, or serum could not replace the effects of the anti-TGF-beta 1 antibody. While these data offer one possible explanation for reports on the inability of SF to stimulate HSC proliferation, they present the basis for a novel model of the regulation of HSC activation wherein: 1) close-range interactions of HSCs with mesenchymal stromal cells do not exclusively determine maintenance of HSC quiescence; 2) competence acquisition by dormant HSCs may involve the down-regulation or inactivation of autocrine TGF-beta 1; and 3) SF may act as a primary growth factor rather than exclusively as a synergistic cytokine.

Loss of antibody binding to prefixed cells: fixation parameters for immunocytochemistry.

The denaturing effects of various types of fixative solutions on 5 cell surface antigens on mouse T-lymphocytes (Thy-1, T-200, Lyt-1, Lyt-2 and Th-B) were studied. For this purpose, cells were fixed with paraformaldehyde, glutaraldehyde, acrolein and osmium tetroxide at various concentrations. Fixed cells were then incubated with monoclonal antibodies and appropriate second stage antibodies or conjugates. The degree of antibody binding to these cells was determined quantitatively using flow-cytometry with a fluorescence-activated cell sorter or with a semi-automatic micro-ELISA system. The data obtained indicate that paraformaldehyde and glutaraldehyde preserve all five tested antigen molecules, whereas antibody binding to cells fixed in acrolein and osmium tetroxide is rapidly reduced at increasing concentrations of the fixative. The optimal concentration of paraformaldehyde is in the range 0.5-1%, whereas glutaraldehyde should be used at concentrations between 0.05 and 0.1%. Cells fixed with 0.5% paraformaldehyde or with 0.05% glutaraldehyde are stable and can be stored for at least one week prior to incubation with antibodies.

An automatic fluorescence micro-ELISA system for quantitative screening of hybridoma supernatants using a protein-A-beta-galactosidase conjugate.

We report on a rapid micro-ELISA screening procedure for the detection of monoclonal antibodies directed against cell surface determinants on lymphoid cells of the mouse. This method employs Terasaki-type trays coated with a monolayer of target cells fixed with 0.02% glutaraldehyde. Cells are incubated with monoclonal antibodies, followed by affinity column-purified rabbit-anti-rat immunoglobulin antibodies and a protein-A-beta-galactosidase conjugate. Binding of antibodies to the cells is visualized by incubation with the substrate 4-methylumbelliferyl galactoside. Fluorescence in the individual wells of the Terasaki trays is then quantitatively analysed within 120 s using a scanning inverted microfluorometer, connected to a digital voltmeter and a desk-top calculator.

Relative stability of inductive properties versus adaptable support capacity for hemopoietic colony formation in the spleen.

The capacity of stromal cells in heterotopic spleen implants to influence colony formation by injected CFU was studied after recovery of host mice from anemia, hypertransfusion or irradiation. The effect of irradiation on the prospective spleen implants was also studied. The ability of implanted spleens to attract CFU and support their colony formation was enhanced following post-implantation recovery in splenectomized, bled or irradiated hosts, whereas prior irradiation of the implant donors with 500 rad x-irradiation impaired these properties. Although slight changes were noted in the ratio of developing erythrocytic and granulocytic colonies, the characteristic inductive properties of the splenic HIM were not considerably changed following post-implantation recovery in non-perturbed or perturbed hosts. A period of anemia did not change the inductive properties and the extent of colony formation of in situ spleens, as measured after the mice had normalized their hematocrits. In contrast, in hypertransfused host mice, hyperplasia and increased colony formation was observed 2 weeks after mice regained their normal hematocrit values, but here also no shifts in E/G ratios were observed. The observations indicate that the characteristic inductive properties of the splenic stroma are quite stable. However, the capacity to attract injected CFU and initiate their colony formation can be manipulated to a great extent during a phase of splenic growth; also the induced changes remain established for a considerable period.

Colony formation by bone marrow cells after incubation with neuraminidase. II. Sensitivity of erythroid progenitor cells for burst promoting activity and erythropoietin and restoration of reduced spleen colony formation in mice pretreated with desialated erythrocyte membrane fragments.

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.

Colony formation by bone marrow cells after incubation with neuraminidase. III. Cell surface repair, homing and growth characteristics of colony forming cells in vivo.

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.

Particle-induced erythropoietin-independent effects of erythroid precursor cells in murine bone marrow.

A possible regulatory action of phagocytic cells on erythropoiesis was investigated by infusion of inert polystyrene latex particles (LAT). LAT appeared to induce changes in the femoral content of erythroid progenitor cells. These changes were most pronounced in primitive erythroid progenitor cells (BFUe) and appeared to be gradually damped in more differentiated populations (CFUe and erythroblasts). LAT did not influence granulocyte/macrophage progenitor cells (CFUc). The effects of LAT could not be attributed to changes in the systemic erythropoietin (EP) concentration. Administration of dexamethason nullified the effect of low doses of LAT, suggesting that phagocytosis of the particles is essential to the observed effects. Erythroid burst formation was previously found to be dependent on a bone marrow associated activity, termed BFA (burst feeder activity). BFA acts as an in vitro inducer of EP-responsiveness in BFUe. In this study it was found that LAT-induced changes in femoral erythroid progenitor cell content were characteristically preceded by corresponding changes in BFA. It was concluded that BFA-associated cells probably play a role in vivo in the early differentiation of erythroid progenitor cells. The present data are interpreted as direct in vivo evidence supporting a two-step regulatory model operating in erythropoiesis and provide evidence that phagocytic cells are a component of the erythroid haemopoietic inductive micro-environment.

Studies of the hemopoietic microenvironments: effects of acid mucopolysaccharides and dextran sulphate on erythroid and granuloid differentiation in vitro.

The effects of acid mucopolysaccharides (AMPS) on in vitro erythrocytic and granulocytic colony formation of murine bone marrow cells have been studied. High concentrations of chondroitin sulphate A, B and C and heparitin sulphate partly or completely inhibited the response of CFU-E to erythropoietin stimulation whereas addition of heparin, hyalyronic acid and keratan sulphate II in concentrations up to 100 microgram/ml did not elicit an inhibition of erythrocytic colony formation. The granulocytic colony formation was not significantly affected by AMPS-addition under these circumstances. Low concentrations of chondroitin sulphate A and B evoked a stimulatory effect on the CFU-E number. The synthetic polyanion dextran sulphate did not affect the erythrocytic and granulocytic colony formation. It is concluded that AMPS can affect the in vitro erythrocytic proliferation and differentiation in concentrations which do not affect the granulocytic maturation. Since stromal cells, i.e. macrophages and reticular cells, in bone marrow in vivo have the ability to produce and remove AMPS in the extravascular matrix we postulate that stomal cells may be involved in the regulation of erythroid progenitor cell maturation.

Kinetics of erythropoiesis in the liver induced in adult mice by phenylhydrazine.

Phenylhydrazine treatment of normal mice elicited a rise in the numbers of CFU-S in blood, spleen and liver. High numbers of CFU-S were found in blood and liver 4 d after the first phenylhydrazine injection. CFU-S in the liver decreased slowly and were absent after 2 weeks. Blood CFU-S returned to normal levels by day 6, whereas spleen CFU-S numbers remained high upto day 12 with a 20-fold increase being apparent between days 5 and 8. Bone marrow CFU-S numbers were relatively unaffected except for a dip between days 4 and 7 with a nadir at day 5 where numbers decreased to 50% of the control levels. Approximately 40% of liver, spleen and blood CFU-S present on the 4th d after initiation of phenylhydrazine treatment, were killed with a single dose of hydroxyurea whereas bone marrow CFU-S numbers were not significantly reduced by the drug. Splenectomy performed before (21 d) or during phenylhydrazine treatment did not diminish the number of CFU-S found in theliver on day 4. A 3 d interval was observed between peak numbers of CFS-U and erythroblasts in the liver which suggests that hepatic CFU-S are able to undergo differentiation along the erythroid pathway. The presence of maceophages was correlated with that of erythroblasts in the hepatic central veins. These macrophages may be essential to the liver environment for induction of erythropoiesis.

Morphological investigation on phenylhydrazine-induced erythropoiesis in the adult mouse liver.

In adult mice suffering from a phenylhydrazine (PHZ)-induced hemolytic anemia, erythropoietic islands were observed in the liver. These islands were studied with the light and electron microscope. Within two days after the beginning of four daily injections of PHZ, erythoid elements appeared in the sinusoids and central veins. A maximum number of erythroblasts was found on day 7. Light and electron microscopic observations revealed that the erythropoietic islands consisted of centrally located macrophages(CM) with a Kupffer cell-like morphology, surrounded by erythroblasts, which were often of the same maturation stage. CM in central veins (CM-V) and in sinusoids (CM-S) were found to have a different morphology. The CM-V phagocytized less circulating red blood cells and were in contact with a smaller number of erythroblasts. Furthermore, the contact areas between erythroblasts and CM-S extended for a much longer distance than those between erythroblasts and CM-V. The progenitor cell for the CM-V is most likely a monocyte, since cells which were morphologically determined as monocytes were found to appear on the first day of the PHZ treatment and differentiated into macrophages within about 2 days. The origin of the CM-S population was less clear, but could be monocytic as well. These data are tentatively explained as a migration of a progenitor of a cellular component of the erythroid micro-environment into the liver after appropriate stimuli. In contrast to fetal liver erythropoiesis, erythroblasts in the adult liver occurred only incidentally extrasinusoidally. Furthermore, specialized membrane contacts between erythroblasts and CM or hepatocytes could not be observed in adult liver. Ferritin could not be detected on the erythroid cell membrane or located in coated vesicles. Also, no ferritin could be observed within or attached to the finger-like processes of CM. The observations suggest that the coated vesicles in erythoid elements are partly exocytotic vesicles and are not specific for ferritin transport. The morphological aspects of PHZ-induced extramedullary erythropoiesis is discussed in relation to the hemopoietic microenvironment.

The T cell-dependent period of the immune response to sheep erythrocytes.

To study the T cell-dependent period of the immune response of mouse spleen cells to sheep erythrocytes the co-operation between T and B cells was abrogated at different times during the in vivo or the in vitro response. The abrogation was performed by killing the T cells with anti-theta serum or anti-H-2 serum. The surviving cells were subsequently cultured in vitro and the number of IgM plaque-forming cells was determined each day. The results indicate that T cells play an important role during the first 3 days of the response in vivo. However, during the in vitro response the presence of the T cells is only required during the first 2 days. The difference between the response in vivo and in vitro is probably due to a synchronous start of the plasma cell development in vitro and a more asynchronous start of this process in vivo.

The recovery of the B-cell population in adult thymectomized, lethally irradiated and bone marrow-reconstituted mice.

The recovery of the B-cell population in adult thymectomized, irradiated and bone marrow-reconstituted mice (T X BM mice was estimated at various times after bone marrow transplantation. The spleen cells to be tested were mixed with dexamethasone-resistant thymocytes (DRT) and sheep red blood cells (SRBC) and transferrred to irradiated recipients. The number of plaque-forming cells (PFC) in the spleen of the recipients was determined 7 days later. Using this functional B-cell assay a sequential appearance of the precursors of IgM-, IgG- and IgA-PFC in the spleen of T X BM mice was observed. The precursors of IgM-PFG (IgM-B cells) were present immediately after transplantation. The first IgG-B cells could be detected at 13-16 days after transplantation and the IgA-B cells finally appeared at 22 days after transplantation. The number of B cells reached a constant and normal level at 30 days after transplantation. The IgM-, IgG- and IgA-B cell development in sham-thymectomized, irradiated and bone narrow-reconstituted mice (ST X BM mice) was virtually the same as in T X BM mice.