CD34+/CD33- cells reselected from macrophage inflammatory protein 1 alpha+interleukin-3--supplemented "stroma-noncontact" cultures are highly enriched for long-term bone marrow culture initiating cells

Human hematopoietic stem cells are thought to express the CD34 stem cell antigen, low numbers of HLA-DR and Thy1 antigens, but no lineage commitment antigens, CD38, or CD45RA antigens. However, fluorescence- activated cell sorted CD34+ subpopulations contain not more than 1% to 5% primitive progenitors capable of initiating and sustaining growth in long-term bone marrow culture initiating cells (LTBMC-ICs). We have recently shown that culture of fresh human marrow CD34+/HLA-DR- cells separated from a stromal layer by a microporous membrane ("stroma- noncontact" culture) results in the maintenance of 40% of LTBMC-ICs. We hypothesized that reselection of CD34+ subpopulations still present after several weeks in stroma-noncontact cultures may result in the selection of cells more highly enriched for human LTBMC-ICs. Fresh marrow CD34+/HLA-DR- cells were cultured for 2 to 3 weeks in stroma- noncontact cultures. Cultured progeny was then sorted on the basis of CD34, HLA-DR, or CD33 antigen expression, and sorted cells evaluated for the presence of LTBMC-ICs by limiting dilution analysis. We show that (1) LTBMC-ICs are four times more frequent in cultured CD34+/HLA- DR- cells (4.6% +/- 1.7%) than in cultured CD34+/HLA-DR- cells (1.3% +/- 0.4%). This suggests that HLA-DR antigen expression may depend on the activation status of primitive cells rather than their lineage commitment. We then sorted cultured cells on the basis of the myeloid commitment antigen, CD33. (2) These studies show that cultured CD34+/CD33- cells contain 4% to 8% LTBMC-ICs, whereas cultured CD34+/CD33+bright cells contain only 0.1% +/- 0.03% LTBMC-ICs. Because LTBMC-ICs are maintained significantly better in stroma-noncontact cultures supplemented with macrophage inflammatory protein 1 alpha (MIP- 1 alpha) and interleukin-3 (IL-3) (Verfaillie et al, J Exp Med 179:643, 1994), we evaluated the frequency of LTBMC-ICs in CD34+/CD33- cells present in such cultures. (3) CD34+/CD33- cells present in MIP-1 alpha + IL-3-supplemented cultures contain up to 30% LTBMC-ICs. The increased frequency of LTBMC-ICs in cultured CD34+ subpopulations may be the result of terminal differentiation of less primitive progenitors, loss of cells that fail to respond to the culture conditions or recruitment of quiescent LTBMC-ICs. The capability to select progenitor populations containing up to 30% LTBMC-ICs should prove useful in studies examining the growth requirements, self-renewal, and multilineage differentiation capacity of human hematopoietic stem cells at the single-cell level.     

Distinct requirements for optimal growth and In vitro expansion of human CD34(+)CD38(-) bone marrow long-term culture-initiating cells (LTC-IC), extended LTC-IC, and murine in vivo long-term reconstituting stem cells

Recently, primitive human bone marrow (BM) progenitors supporting hematopoiesis in extended (>60 days) long-term BM cultures were identified. Such extended long-term culture-initiating cells (ELTC-IC) are of the CD34(+)CD38(-) phenotype, are quiescent, and are difficult to recruit into proliferation, implicating ELTC-IC as the most primitive human progenitor cells detectable in vitro. However, it remains to be established whether ELTC-IC can proliferate and potentially expand in response to early acting cytokines. Here, CD34(+)CD38(-) BM ELTC-IC (12-week) were efficiently recruited into proliferation and expanded in vitro in response to early acting cytokines, but conditions for expansion of ELTC-IC activity were distinct from those of traditional (5-week) LTC-IC and murine long-term repopulating cells. Whereas c-kit ligand (KL), interleukin-3 (IL-3), and IL-6 promoted proliferation and maintenance or expansion of murine long-term reconstituting activity and human LTC-IC, they dramatically depleted ELTC-IC activity. In contrast, KL, flt3 ligand (FL), and megakaryocyte growth and development factor (MGDF) (and KL + FL + IL-3) expanded murine long-term reconstituting activity as well as human LTC-IC and ELTC-IC. Expansion of LTC-IC was most optimal after 7 days of culture, whereas optimal expansion of ELTC-IC activity required 12 days, most likely reflecting the delayed recruitment of quiescent CD34(+)CD38(-) progenitors. The need for high concentrations of KL, FL, and MGDF (250 ng/mL each) and serum-free conditions was more critical for expansion of ELTC-IC than of LTC-IC. The distinct requirements for expansion of ELTC-IC activity when compared with traditional LTC-IC suggest that the ELTC-IC could prove more reliable as a predictor for true human stem cell activity after in vitro stem cell manipulation.     

Direct contact between CD34+lin- cells and stroma induces a soluble activity that specifically increases primitive hematopoietic cell production

Perfused human bone marrow (BM) mononuclear cell (MNC) cultures result in a greater long-term culture-initiating cell (LTC-IC) output than parallel CD34+lin- cell cultures, even when CD34+lin- cells are placed on irradiated preformed stroma (IPFS). This difference has been attributed to accessory cell effects that are potentiated by medium perfusion. The present study investigated the relative contributions of direct contact- and soluble-mediated mechanisms of accessory cells in this culture system. CD34+lin- cells within (i.e., in contact with) the MNC accessory cell mixture generated greater LTC-IC output than CD34+lin- cells in contact with IPFS. Incubation of CD34+lin- cells with MNC conditioned medium (CM) resulted in partial restoration of MNC accessory activity, while CM from IPFS had no activity on LTC-IC output. Interestingly, the level of LTC-IC output supported by MNC CM was equivalent to that supported by direct contact with IPFS. CD34+lin- cells were then cultured in Transwell inserts either alone, with IPFS (direct contact), or with IPFS below the insert. Direct contact with IPFS significantly increased the output of cells, CFU-GM, and LTC-IC from CD34+lin- cells. IPFS below the insert also resulted in significantly increased cell and CFU-GM output, but did not significantly affect LTC-IC output. Further experiments using CM from CD34+lin- cells and IPFS cultures showed that LTC-IC supportive activity was present only when direct contact was allowed between CD34+lin- cells and IPFS. ELISA and RT-PCR experiments showed that contact did not induce changes in the levels of several known growth factors, including GM-CSF, IL-1beta, IL-3, IL-6, IL-11, LIF, KL, FL, Tpo, TGF-beta, and MIP-1alpha. These results indicate that direct contact between CD34+lin- cells and IPFS induces soluble activity, which specifically increases LTC-IC output from CD34+lin- cell cultures, providing evidence for a novel direct contact-mediated two-way mechanism of communication between primitive hematopoietic cells and stroma.     

Phenotypic and functional characterization of long-term culture- initiating cells present in peripheral blood progenitor collections of normal donors treated with granulocyte colony-stimulating factor

Granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood progenitor cells (PBPC) have successfully been used as stem cells for both autologous and allogeneic transplants. However, little is known concerning the absolute number and phenotype of primitive progenitors, such as long-term culture-initiating cells (LTC-IC) in mobilized PBPC. The aim of our study was to evaluate the capacity of G- CSF to mobilize LTC-IC in the PB of normal individuals and to evaluate the phenotypic and functional characteristics of G-CSF mobilized LTC- IC. G-CSF was administered to 29 healthy volunteers at 7.5 micrograms or 10 micrograms/kg/d subcutaneously (SC) for 5 consecutive days and PBPC were harvested on day 6. Mobilization with G-CSF increased the absolute number of week 5 LTC-IC in PB 60-fold, while the number of CD34+ cells and committed colony forming cells (CFC) was increased sevenfold to 12-fold. The frequency of CFC and week 5 LTC-IC in CD34+ cells selected by fluorescence-activated cell sorter (FACS) from mobilized PBPC was 2 +/- 0.3-fold and 9 +/- 2.2-fold higher respectively than in CD34+ cells selected from unmobilized PBMNC. CFC were enriched in the CD34+ CD38+ and CD34+ HLA-DR+ populations. The absolute number of LTC-IC present in CD34+ CD38- and CD34+ HLA-DR- cells selected by FACS from either mobilized PBPC, unmobilized PBMNC or steady state bone marrow (BM) was similar (0.5% to 2%). In contrast to unmobilized PBMNC or steady state BM CD34+ CD38+ and CD34+ HLA-DR+ cells, which contain less than 0.1% LTC-IC, CD34+ CD38+ and CD34+ HLA- DR+ cells sorted from mobilized PBPC contained 0.5% to 5% of cells capable of sustaining hematopoiesis in long-term cultures for 5 weeks. However, 90% to 95% of LTC-IC present in mobilized CD34+ CD38+ and CD34+ HLA-DR+ cells were not able to sustain hematopoiesis for 8 weeks, while 30% of CD34+ CD38- and CD34+ HLA-DR- LTC-IC present in mobilized PBPC could sustain hematopoiesis for at least 8 weeks. This suggests that the majority of CD34+ CD38+ and CD34+ HLA-DR+ week 5 LTC-IC represent progenitors at an intermediate state of differentiation. We conclude that G-CSF effectively mobilizes LTC-IC in the blood of normal individuals. Although a fraction of these cells has functional characteristics similar to those of steady state PBMNC or BM LTC-IC, more than 85% of mobilized PBPC LTC-IC are CD34+ CD38+ and CD34+ HLA- DR+, capable of sustaining hematopoiesis for 5 weeks, but not for 8 weeks. The functional and phenotypic characterization of primitive and more mature populations of LTC-IC in mobilized PBPC should prove extremely useful in future studies examining the role of these progenitors in engraftment following transplantation.     

Expansion of granulocyte colony-stimulating factor/chemotherapy-mobilized CD34+ hematopoietic progenitors: role of granulocyte-macrophage colony-stimulating factor/erythropoietin hybrid protein (MEN11303) and interleukin-15

Ex vivo stroma-free static liquid cultures of granulocyte colony-stimulating factor (G-CSF)/chemotherapy-mobilized CD34+ cells were established from patients with epithelial solid tumors. Different culture conditions were generated by adding G-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF), Flt3 ligand (Flt3), megakaryocyte growth and development factor (Peg-rHuMGDF), GM-CSF/erythropoietin (EPO) hybrid protein (MEN11303), and interleukin-15 (IL-15) to the basic stem cell factor (SCF) + interleukin-3 (IL-3) + EPO combination. This study showed that, among the nine different combinations tested in our 5% autologous plasma-containing cultures, only those containing IL-3/SCF/Flt3/MEN11303 and IL-3/SCF/Flt3/MEN11303/IL-15 significantly expanded colony-forming unit granulocyte-macrophage (CFU-GM), burst-forming unit erythroid (BFU-E), long-term culture-initiating cells (LTC-IC), CD34+, and CD34+/CD38- cells after 14 days of culture. Particularly, the addition of IL-15 to IL-3/SCF/Flt3/MEN11303 combination produced a significant increase of LTC-IC, with an average 26-fold amplification as compared to input cells, without any detrimental effect on CFU-GM and BFU-E expansion. This combination also produced a statistically significant 3.6-fold expansion of primitive CD34+/CD38- cells. Moreover, this study confirms the previously described erythropoietic effect of MEN11303, which, in our experience, was the only factor capable of expanding BFU-E. Compared to equimolar concentrations of GM-CSF and EPO, MEN11303 hybrid protein showed a significantly higher capacity of expanding CFU-GM, BFU-E, LTC-IC, CD34+, and CD34+/CD38- cells when these cytokines were tested in combination with IL-3/SCF/Flt3. These cultures indicated that Peg-rHuMGDF addition to IL-3/SCF/EPO/Flt3 does not affect CFU-GM and BFU-E expansion but, unlike G-CSF or GM-CSF, it does not decrease the ability of Flt3 to expand primitive LTC-IC. These studies indicate that, starting from G-CSF/chemotherapy-mobilized CD34+ cells, concomitant expansion of primitive LTC-IC, CFU-GM, BFU-E, CD34+, and CD34+/CD38- cells is feasible in simple stroma-free static liquid cultures, provided IL-3/SCF/Flt3/MEN11303/IL-15 combination is used as expanding cocktail in the presence of 5% autologous plasma.     

Chronic myelogenous leukemia primitive hematopoietic progenitors demonstrate increased sensitivity to growth factor-induced proliferation and maturation

We investigated whether primary chronic myelogenous leukemia (CML) hematopoietic progenitors demonstrated altered proliferation and maturation in response to growth factor (GF) stimulation.The effect of GF stimulation on proliferation and expansion of committed and primitive progenitors (colony forming cells [CFC]) was evaluated. Culture of CML and normal CD34(+) cells with different GF for 7 days resulted in similar expansion of committed progenitors (CFC). In contrast, GF culture conditions that expanded normal primitive progenitors (week-6 long-term culture-initiating cells (LTC-IC)] led to depletion of CML LTC-IC numbers. GF culture also resulted in increased depletion of week-10 extended LTC-IC, which represent an even more primitive progenitor population, from CML compared with normal CD34(+) cells. CML CD34(+) cells enter into cycle more quickly than normal CD34(+) cells and CML CFC expansion was accelerated compared to normal CFC. Evaluation of primitive progenitor proliferation using PKH-26 and single-cell LTC-IC analysis demonstrated that the majority of CML LTC-IC remaining after GF culture originated from divided CD34(+) cells, whereas GF-cultured normal LTC-IC were derived mainly from undivided cells. Depletion of CML primitive progenitor numbers in association with increased proliferation suggests increased sensitivity to GF-induced maturation.These studies indicate that CML primitive progenitors have enhanced sensitivity to GF-induced cell division and maturation. Altered GF responsiveness may contribute to abnormal expansion of malignant myeloid cells in CML. These findings may also be applied toward the development of novel approaches to select benign stem cells in CML.     

Stromal fibroblast heparan sulfate is required for cytokine-mediated ex vivo maintenance of human long-term culture-initiating cells

We have recently demonstrated that 50% of primitive human long-term culture-initiating cells (LTC-IC) are maintained for up to 8 weeks in stroma-dependent cultures in which progenitor-stroma contact is prevented (stroma noncontact), or when progenitors are cultured in medium conditioned by stromal feeders. This indicates that factors responsible for LTC-IC maintenance are present in soluble form in stromal supernatant (SN). Although the picogram concentrations of cytokines present in stromal SN can induce the differentiation of CD34+/HLA-DR- (DR-) cells to clonogenic cells (colony forming cells; CFC), they maintain only 10% of LTC-IC for 5 weeks, suggesting that factors other than these cytokines are required for LTC-IC maintenance. To characterize the factor(s) in stromal SN responsible for LTC-IC maintenance, we purified glycoproteins and proteoglycans (PG) from the SN of the LTC-IC supportive murine marrow stromal fibroblast cell line M2-10B4 by ion exchange high performance liquid chromatography (HPLC). Culture of DR- cells in a combination of M2-10B4-derived PG, but not glycoproteins and picogram concentrations of recombinant human interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), leukemia inhibitory factor (LIF), granulocyte- macrophage colony-stimulating factor (GM-CSF), and macrophage inflammatory protein-1alpha (MIP-1alpha) resulted in the recovery of 96% +/- 8% of LTC-IC maintained in cultures supplemented with unfractionated stromal SN. LTC-IC maintenance was largely retained after digestion of the PG-rich fraction with proteinase K and after dissociative gel filtration chromatography, but was completely abolished following treatment with nitrous acid, which digests heparan sulfate glycosaminoglycans (HS GAG). As for M2-10B4-derived HS GAG, high concentrations of bovine kidney HS GAG, but not bovine tracheal chondroitin sulfate, significantly improved cytokine-mediated LTC-IC maintenance. Maintenance of LTC-IC by these nonmarrow-derived HS GAG was, however, significantly lower than that seen with M2-10B4-derived HS. These studies demonstrate a role for marrow stroma-derived HS GAG in the long-term in vitro maintenance of human LTC-IC. Further structure-function analysis of these HS GAG may have important implications for ex vivo stem cell expansion and gene transfer into hematopoietic progenitors.     

Human LTC-IC can be maintained for at least 5 weeks in vitro when interleukin-3 and a single chemokine are combined with O-sulfated heparan sulfates: requirement for optimal binding interactions of heparan sulfate with early-acting cytokines and matrix proteins

We have shown that stromal O-sulfated heparan sulfate glycosaminoglycans (O-S-GAGs) regulate primitive human hematopoietic progenitor cell (HPC) growth and differentiation by colocalizing heparin-binding cytokines and matrix proteins with HPC in stem cell "niches" in the marrow microenvironment. We now show that long-term culture-initiating cells (LTC-IC) are maintained for 5 weeks in the absence of stroma when O-S-GAGs are added to IL-3 and either MIP-1alpha or PF4 (LTC-IC maintenance without GAGs, 32 +/- 2%; with GAGs, 95 +/- 7%; P <.001). When cultured with 5 additional cytokines, O-S-GAGs, IL-3, and MIP-1alpha, LTC-IC expanded 2- to 4-fold at 2 weeks, and 92 +/- 8% LTC-IC were maintained at 5 weeks. Similar results were seen when PF4 replaced MIP-1alpha. Although O-S-GAG omission did not affect 2-week expansion, only 20% LTC-IC were maintained for 5 weeks. When O-S-heparin was replaced by completely desulfated-, N-sulfated (O-desulfated), or unmodified heparins, LTC-IC maintenance at week 5 was not better than with cytokines alone. Unmodified- and O-S-heparin, but not desulfated- or N-sulfated heparin, bound to MIP-1alpha, IL-3, PF4, VEGF, thrombospondin, and fibronectin. However, the affinity of heparin for thrombospondin and PF4, and the association and dissociation rates of heparin for PF4, were higher than those of O-S-heparin. We conclude that (i) although cytokines may suffice to induce early expansion, adult human LTC-IC maintenance for longer than 1 month requires O-S-GAGs, and (ii) HPC support may depend not only on the ability of GAGs to bind proteins, but also on optimal affinity and kinetics of interactions that affect presentation of proteins in a biologically active manner to progenitors. (Blood. 2000;95:147-155)     

Role of cytokines in sustaining long-term human megakaryocytopoiesis in vitro.

An in vitro liquid suspension culture system was used to determine the role of cytokines in sustaining long-term human megakaryocytopoiesis. Bone marrow cells expressing CD34 but not HLA-DR (CD34+DR-) were used as the inoculum of cells to initiate long-term bone marrow cultures (LTBMC). CD34+DR- cells (5 x 10(3)/mL) initially contained 0.0 +/- 0.0 assayable colony-forming unit-megakaryocytes (CFU-MK), 6.2 +/- 0.4 assayable burst-forming unit-megakaryocytes (BFU-MK), and 0.0 +/- 0.0 megakaryocytes (MK). LTBMCs were recharged every 48 hours with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1 alpha (IL-1 alpha), IL-3, and/or IL-6, alone or in combination. LTBMCs were demidepopulated weekly or biweekly, the number of cells and MK enumerated, and then assayed for CFU-MK and BFU-MK. LTBMCs receiving no cytokine(s) contained no assayable CFU-MK or BFU-MK and no observable MK. LTBMCs receiving GM-CSF, IL-1 alpha, and/or IL-3 contained assayable CFU-MK and MK but no BFU-MK for 10 weeks of culture. The effects of GM-CSF and IL-3, IL-1 alpha and IL-3, but not GM-CSF and IL-1 alpha were additive with regards to their ability to augment the numbers of assayable CFU-MK during LTBMC. LTBMCs supplemented with IL-6 contained modest numbers of assayable CFU-MK for only 4 weeks; this effect was not additive to that of GM-CSF, IL-1 alpha, or IL-3. The addition of GM-CSF, IL-1 alpha, and IL-3 alone or in combination each led to the appearance of significant numbers of MKs during LTBMC. By contrast, IL-6 supplemented cultures contained relatively few MK. These studies suggest that CD34+DR- cells are capable of initiating long-term megakaryocytopoiesis in vitro and that a hierarchy of cytokines exists capable of sustaining this process.     

Human cytomegalovirus suppression of and latency in early hematopoietic progenitor cells

Bone marrow cells (BMC) are involved in the pathogenesis of human cytomegalovirus++ (HCMV) infections, and the hematopoietic cells are probable sites of HCMV latency in healthy donors. In vitro studies have indicated both a direct inhibitory effect of HCMV on proliferation and differentiation of myeloid bone marrow progenitors and an impairment of bone marrow stroma cell function by HCMV. The purpose of the present study was to establish whether the suppressing effect could be limited to subsets of immature CD34+ BMC and to investigate the role of immature cell populations as possible sites of HCMV latency. CD34+ cells from healthy HCMV-seropositive and -seronegative donors were sorted according to the expression of HLA-DR (CD34+ HLA-DR+ and CD34+ HLA-DR- cells). The progenitor growth of hematopoietic progenitor cells from seronegative donors was examined by colony and single-cell assays after in vitro infection with HCMV. To determine the susceptibility of the CD34+ cells to HCMV infection in vitro and in vivo, cells of both subsets from seronegative and seropositive donors were analyzed for the presence of HCMV DNA by polymerase chain reaction. HCMV infection in vitro inhibited the interleukin-1alpha (IL-1alpha)-, IL-3-, granulocyte colony-stimulating factor-, granulocyte-macrophage colony-stimulating factor-, and stem cell factor-induced proliferation in single-cell assays of CD34+ HLA-DR- cells by 34%. In contrast, the colony growth of the CD34+ HLA-DR+ subset was suppressed in cells from only 3 of the 8 donors. However, in vitro HCMV infection of the CD34+ HLA-DR+ progenitor cells inhibited the proliferation of all donors tested when hematopoietic growth factors were used individually to promote progenitor growth. In addition, the formation of burst-forming units- erythroid and colony-forming units-granulocyte, erythrocyte, monocyte, megakaryocyte was reduced 40% to 60% by HCMV in vitro. In contrast, the growth of high proliferative potential colony-forming cells was not inhibited after in vitro HCMV infection. Furthermore, HCMV DNA was detected in both CD34+ HLA-DR- and CD34+ HLA-DR+ progenitors from in vitro-infected HCMV-seronegative donors and cells from HCMV- seropositive donors. Taken together, the early progenitors defined as CD34+ HLA-DR- and CD34+ HLA-DR+ are directly suppressed in their proliferation by HCMV in vitro, and hematopoietic stem cells are also sites of HCMV latency in healthy HCMV-seropositive donors.     

Optimization of culture conditions to enhance transfection of human CD34+ cells by electroporation

The ability to culture CD34+ stem cells, while maintaining their pluripotency, is essential for manipulations such as gene transfection for therapeutic trials. Human peripheral blood (PB) CD34+ cells (> or = 90% purity) were cultured for up to 4 days in serum-free culture medium supplemented with thrombopoietin (TPO), stem cell factor (SCF), Flt-3 ligand (Flt-3L), with or without PIXY321 (IL-3/GM-CSF fusion protein) and human serum. The CD34 mean fluorescence intensity (MFI) and cell cycle status were evaluated daily using flow cytometry and hypotonic propidium iodide. Prior to culture (day 0), 97.0 +/- 0.9%, 1.9 +/- 0.3% and 1.0 +/- 0.6% of the selected CD34+ cells were in G0-G1, S-phase, or G2-M, respectively. After 2-4 days in culture with TPO/SCF/Flt-3L, there was an increase in the percent of cells in S-phase to 26.4 +/- 0.1% without significant loss of CD34 MFI. The addition of PIXY321 increased.the percentage of CD34+ cells in S-phase to 36.3 +/- 4.0%, but the CD34 MFI and numbers of CFU (colony-forming units) were significantly decreased at day 3 when cultured with PIXY321 or various recombinant cytokine combinations that included IL-3 and IL-6. There is an increase from day 0 to day 4 in the percentages of CD34+ with CD38-, HLA-DR-, and c-kit(low), but not Thy-1+ cells. Electroporation with EGFP reporter gene showed that 1-2 days of pre-stimulation in X-VIVO 10 supplemented with TPO/SCF/Flt-3L was necessary and sufficient for efficient transfection. Flow cytometry analysis demonstrated that 22% of the viable cells are CD34+/EGFP+ 48 h post electroporation. The introduced reporter gene appears to be stable as determined by EGFP+/LTC-IC (long-term colony-initiating cells), at 30-40 positive colonies (16 +/- 7%) per 1 x 10(5) electroporated CD34+ cells.     

Long-term culture-initiating cell expansion is dependent on frequent medium exchange combined with stromal and other accessory cell effects

Despite considerable effort, the expansion of long-term culture- initiating cells (LTC-ICs) in cultures of purified hematopoietic cells has not yet been achieved. In contrast, LTC-IC expansion has been attained in cultures of bone marrow mononuclear cells (MNC) using frequent medium exchange. The use of frequent medium exchange was, therefore, examined in cultures of CD34-enriched cells. In stromal- free, CD34-enriched cell cultures, medium exchange intervals ranging from 2 days to no feeding for 14 days gave similar results. Six different growth factor combinations, reported by other groups to give optimal expansion of CD34-enriched cells, were tested in comparison with the control combination of IL-3/GM-CSF/Epo/SCF. None of the combinations resulted in improved colony-forming unit-granulocyte macrophage (CFU-GM) expansion or LTC-IC maintenance, although two were equivalent. All stromal-free cultures resulted in loss of LTC-IC to half of input. Because of the limited effect of medium exchange and growth factor variations on CD34-enriched cell cultures, the effect of preformed stroma was next examined. Preformed stroma increased cell (3- fold), CFU-GM (5-fold), and LTC-IC (3-fold) output, but only when the medium was exchanged every other day. Under these conditions, the number of LTC-IC was maintained near input level. The lack of LTC-IC expansion in CD34-enriched cell cultures prompted experiments to examine the effect of cell purification. Parallel cultures were performed at CD34+lin- cell purities of 20%, 40%, 70%, and 95%, with each well containing exactly 4,000 CD34+lin- cells in addition to the CD34- accessory cells required to give the desired percentage. Also, MNC from the same source (approximately 2% CD34+lin-) were cultured at a concentration to give 4,000 CD34+lin- cells per well. As CD34+lin- cell purity was decreased from 95% to 2%, the output of cells, CFU-GM, and LTC-IC increased by threefold to fivefold. The loss of culture performance with purification was likely due to the removal of important accessory cells, because the levels of endogenously produced leukemia inhibitory factor and IL-6 were found to decline significantly with increasing CD34+lin- cell purity. In summary, preformed stroma abrogated the decrease in cell and CFU-GM output from cultured CD34- enriched cells and led to LTC-IC maintenance. In contrast, MNC inocula resulting in a growing stromal layer during the culture led to LTC-IC expansion (3.2-fold).(ABSTRACT TRUNCATED AT 400 WORDS)     

Potentiation of early hematopoiesis by tumor necrosis factor-alpha is followed by inhibition of granulopoietic differentiation and proliferation.

We have previously shown that tumor necrosis factor-alpha (TNF alpha) strongly potentiates interleukin-3 (IL-3)-induced short-term proliferation of human CD34+ hematopoietic progenitor cells (HPC). Using longer term cultures of CD34+ HPC, we demonstrate here that this initial potentiation ceases after 10 to 12 days; whereupon TNF alpha displays inhibitory effects. Thus, TNF alpha was found to inhibit cells of granulocytic affiliation while it potentiates the development of maturing cells of the monocytic lineage both in liquid and semi-solid (day 14 colony-forming unit) cultures. TNF alpha was demonstrated to reversibly block granulocytic differentiation at the level of uncommitted CD13-, CD15- blast cells that accumulate in IL-3 + TNF alpha cultures. Furthermore, growth of committed granulocytes (CD15+) from IL-3 cultures was also inhibited by TNF alpha through an arrest of cell cycle in G0/G1. Finally, the use of neutralizing anti-TNF alpha monoclonal antibody and limiting dilution studies indicate that the inhibitory effects of TNF alpha are direct. Taken together, our data demonstrate that, following a phase of potentiation of proliferation of early HPC, TNF alpha displays direct inhibitory effects due to negative interference with both granulocytic differentiation and proliferation of granulocytic cells.     

Different behaviour of fresh and cultured CD34+ cells during immunomagnetic separation

In-vitro expansion of human cord blood (CB) cells could enhance peripheral blood recovery and ensure long-term engraftment of larger recipients in the clinical transplant setting. Enrichment of CD34+ cells using the MiniMACS column has been evaluated for the preparation of CB CD34+ cells before and after expansion culture. Repurification of CD34+ cells after culture would assist accurate phenotypic and functional analysis. When fresh CB mononuclear cells (MNC) were separated, the MACS positive (CD34+) fraction (90.1% pure) contained a mean (+/- SD, n = 5) of 93.0 +/- 8.0% of the eluted CD34+ cells, 99.6 +/- 0.7% of the CFU-GM and all of the eluted long-term culture-initiating cells (LTC-IC). Cord blood CD34+ cells were then cultured for 14 d with IL-3, IL-6, SCF, G-CSF and GM-CSF, each at 10 ng/ml. The total cell expansion was 2490 +/- 200-fold and the CD34+ cell expansion was 49 +/- 17-fold. The percentage of CD34+ cells present after expansion culture was 1.2 +/- 0.85%. When these cells were repurified on the MiniMACS column, the MACS positive fraction only contained 40.3 +/- 13.4% of the eluted CD34+ cells which was enriched for the mature CD34+ CD38+ subset, 24.4 +/- 8.8% of the eluted CFU-GM and 79.5 +/- 11.0% of the LTC-IC. The remaining cells were eluted in the MACS negative fraction. In conclusion, repurification of cultured CD34+ cells does not yield a representative population and many progenitors are lost in the MACS negative fraction. This can give misleading phenotypic and functional data. Cell losses may be important in the clinical setting if cultured cells were repurified for purging.     

Effects of CD34+ cell selection and perfusion on ex vivo expansion of peripheral blood mononuclear cells

Ex vivo expansion of peripheral blood mononuclear cells (MNCs), cultured both directly and after selection for CD34+ cells, was compared in static and continuously perfused cultures containing interleukin (IL)-3, IL-6, granulocyte colony-stimulating factor (G- CSF), and stem cell factor (SCF). Cultures inoculated with either MNCs or CD34+ cells produced cells that were remarkably similar after 10 days of culture, as evidence by cell morphology, expression of CD34, CD33, CD15, and CD11b, and the fractions of cells giving rise to colony- forming units granulocyte-monocyte (CFU-GM) and long-term culture- initiating cells (LTC-IC). Static and perfusion cultures gave similar average total cells and CFU-GM expansions for both MNC and CD34+ cell cultures. However, those samples that performed poorly in static culture performed at near-normal levels in perfusion. In addition, perfusion supported higher LTC-IC numbers for both MNC and CD34+ cell cultures. While total cell expansion was about ten times greater in CD34+ cell cultures (approximately 100-fold), CFU-GM expansion (approximately 20-fold) was similar for both MNC and CD34+ cell cultures. The similar distribution of cell types produced in MNC and CD34+ cell cultures allows direct comparison of total and colony- forming cell production. After 15 days in perfusion, MNC cultures produced 1.5-, 2.6-, and 2.1-fold more total cells, CFU-GM, and LTC-IC, respectively, than the same sample selected and cultured as CD34+ cells. Even if the CD34+ selection process was 100% efficient, CFU-GM production would be 1.5-fold greater for MNCs than for CD34+ cells.     

Extended long-term culture reveals a highly quiescent and primitive human hematopoietic progenitor population

Long-term culture-initiating cells (LTC-IC) are hematopoietic progenitors able to generate colony-forming unit-cells (CFU) after 5 to 8 weeks (35 to 60 days) of culture on bone marrow (BM) stroma and represent the most primitive progenitors currently detectable in vitro. We have recently reported that long-term cultures initiated with CD34+CD38- cells from BM or cord blood are able to continue generating CFU for at least 100 days, ie, beyond the standard LTC-IC period. In this report, single-cell cultures from cord blood and retroviral marking of cord blood and BM were used to study whether the subpopulation of CD34+CD38- cells able to generate CFU beyond 60 days ("extended long-term culture-initiating cells" or ELTC-IC) are functionally distinct from LTC-IC in terms of timing of initial clonal proliferation and generative capacity. All cord blood LTC-IC formed clones of greater than 50 cells by day 30. In contrast, cord blood ELTC- IC proliferated later in culture, 50% forming clones after day 30. Although efficient retroviral marking of LTC-IC was seen (25% to 45%), marking of ELTC-IC was inefficient (< 1%), consistent with a more quiescent progenitor population. There was a positive correlation between time of clonal proliferation and generative capacity. ELTC-IC generated threefold to fourfold more progeny than did LTC-IC (P < .002). These studies show that there is a functional hierarchy of progenitors in long-term culture which correlates with their level of quiescence. By extending the LTC-IC assay, a more primitive progenitor may be studied that may be functionally closer to the human long-term repopulation stem cell in vivo.     

Presence of primitive lymphoid progenitors with NK or B potential in ex vivo expanded bone marrow cell cultures

OBJECTIVE: In previous work, we showed that CD34+ bone marrow cells can be successfully expanded along the myeloid pathway in stroma- and serum-free conditions in the presence of SCF+IL-3+IL-6+Flt3-l+G-CSF+MGDF. Due to the lack of phenotypically detectable lymphoid cells, it was necessary to address the question of the lymphoid potential of the expanded populations under these conditions. MATERIALS AND METHODS: The present report describes a long-term culture system that supports human B- and NK-cell differentiation from the day 14 fraction without further selection of the more primitive cells. In NK proliferation assays, the cells were maintained over stroma cells in the presence of IL-2 for 4-5 weeks. NK initiating cells (NK-IC) were determined by a limiting dilution assay. In B-cell cultures, the expanded cells were maintained over MS5 in the presence of Flt3-l for 4-8 weeks. RESULTS: NK cells rose from 0.2%+/-0.04% at culture initiation to 71%+/-6% at week 5. These cells displayed cytolytic activity. NK-IC evaluation showed a mean 18-fold expansion in the day 14 expanded fraction as compared to the initial day 0 fraction. Similarly, CD19+ cells rose from 0.1% at culture initiation to 30%+/-1% at week 6. Cells produced under these B-LTC conditions were CD34-CD19+CD10+. We also demonstrated that the CD34+/Lin- sorted cells from the day 14 fraction gave rise to NK and B cells. CONCLUSION: This culture system permits the revelation of a population that, although poorly represented in terms of phenotypically detectable cells, nevertheless retains high levels of lymphoid NK and B potential after 14 days expansion. Such data suggest the persistence, or expansion, of lymphoid progenitors and, hence, the multipotentiality of the expanded progenitor/stem cells.     

Loss of primitive hematopoietic progenitors in patients with human immunodeficiency virus infection

A number of hematologic abnormalities, including cytopenias, have been observed in patients with human immunodeficiency virus (HIV) infection. To elucidate their mechanisms, primitive cells from bone marrow aspirates of 21 patients with HIV-1 infection were quantitated by flow cytometry. The mean percentage of CD34+ cells is not significantly altered in HIV-1-infected patients in comparison with HIV-1- seronegative controls. In contrast, two- and three-color immunofluorescence analysis showed that in all HIV-1 samples, most CD34+ cells coexpressed the CD38 antigen. The proportion of HIV-1- derived CD34+ cells that did not express the CD38 antigen was significantly lower (HIV-1+: mean, 1.73%; controls: mean, 14%; P < .0005) than in controls. Moreover, of Thy-1+ cells, the proportion of CD34+ cells was twofold lower in HIV-1-infected patients (HIV-1+: mean, 12%; controls, 25%, P < .0005), which suggests that phenotypically primitive cells are depleted in HIV-1 infection. In vitro functional analysis in long-term cultures of sorted CD34+ cells from seven HIV-1 patients showed that CD34+ cells from HIV-1 patients generated much fewer colonies both in the nonadherent and adherent layers than CD34+ cells from controls after 5 weeks of culture (10-fold and four-fold less, respectively). Precise long-term culture initiating cell (LTC-IC) frequency in the CD34+ cell population was determined in three patients by limiting dilution and was markedly decreased in comparison to that of normal controls (from twofold to > sevenfold decreased). To determine if primitive cells were infected by HIV-1, both methylcellulose colonies generated from long-term culture of CD34+ cells and various CD34+ cell fractions purified by flow cytometry were evaluated for the presence of HIV-1 by polymerase chain reaction (PCR). Progeny from long-term culture was HIV-1-negative in three samples. In addition, using a sensitive PCR technique, the HIV-1 genome could not be detected in CD34+, CD34+/CD38-, and CD34+/CD4+ cells. These data show that hematologic disorders in HIV disease may be the consequence of a deficit of primitive cells. However, direct infection of these cells by HIV-1 does not seem to be responsible for this defect.     

Interleukin-7 induces the proliferation of normal human B-cell precursors.

In the present study, we investigated the effects of human recombinant interleukin-7 (IL-7) on the proliferation of enriched hematopoietic cells isolated from human adult and fetal bone marrow (BM). In cultures of CD34+ cells, IL-7 was found to induce dose-dependent incorporation of 3H-thymidine (3H-TdR), but had no demonstrable effect on the development of myeloid colony-forming cells. Numbers of B-cell precursors (BCP), initially present within CD34+ populations and which included a CD34+CD20+ subset, were significantly increased when CD34+ BM cells were cultured in the presence of IL-7. This effect was most striking on CD20+ BCP, and resulted at least partly from higher numbers of cycling cells as indicated by Hoechst 33342 fluorescence (Calbiochem, Behring Diagnostics, La Jolla, CA). These results indicate that IL-7 promotes the growth of BCP within the CD34+ compartment. In line with the B-lineage affiliation of CD34+ target cells, committed BCP (CD10+ CD19+ surface IgM-) isolated from BM were also found to proliferate in response to IL-7. Interestingly, this effect of IL-7 was strongly potentiated by the addition of IL-3. Taken together, and in accordance with previous observations on murine cells, our data indicate that IL-7 acts as a growth factor during the ontogeny of human B lymphocytes.     

Cell cycle distribution of cord blood-derived haematopoietic progenitor cells and their recruitment into the S-phase of the cell cycle

The objective of this study was to evaluate the cycling status of cord blood (CB)-derived colony-forming cells (CFC) and long-term culture-initiating cells (LTC-IC), and their recruitment into the S-phase of the cell cycle. By using the cytosine arabinoside (Ara-C) suicide approach, we found that only small proportions of both CFC and LTC-IC were in the S-phase of the cell cycle. These estimates were confirmed by flow cytometric DNA analysis, which showed that 96 +/- 2% of CB-derived CD34+ cells were in G0/G1 and only 1.6 +/- 0.4% in the S-phase. Staining of CD34+ cells with an antistatin monoclonal antibody, a marker of the G0 phase, indicated that among CD34+ cells with a flow cytometric DNA content typical of the G0/G1 phase 68 +/- 7% of cells were in the G0 phase of the cell cycle. Incubation (24 h) with interleukin 3 (IL-3), recombinant human stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) significantly increased the proportion of cells in the S-phase for both CFC and LTC-IC without inducing any loss in numbers. Flow cytometric DNA analysis also showed an increase in CD34+ cells in the S-phase upon continuous exposure to these cytokines. Our findings indicate that: (i) very few CB-derived CFC or LTC-IC were in the S-phase of the cell cycle; (ii) a substantial amount of CD34+ cells with a flow cytometric DNA content typical of the G0/G1 fraction was cycling, as found in the G1 phase of the cell cycle; and (iii) 24-h incubation with IL-3, SCF and G-CSF could drive a proportion of progenitor cells into the S-phase without reducing their number. These data might be useful for gene transfer protocols and the ex vivo expansion of CB-derived progenitor cells.