Liquid Suspension Culture Research Articles

Rescue of Hematopoietic Stem Cells Following High-Dose Radiation Injury Using Ex Vivo Culture on Endothelial Monolayers

Military personnel are at significant risk for potentially lethal myeloablative injury secondary to nuclear accident, nuclear attack, or chemical weapons attack. In an attempt to develop culture conditions in which hematopoietic stem cells might be rescued from the effects of radiation, we irradiated (1,000 cGy split dose) 6-week-old C57BL/6 (Ly 5.1) and syngeneic C57BL/6 (Ly 5.2) mice and tested whether bone marrow mononuclear cells (MNCs) harvested postirradiation could be rescued via coculture in porcine microvascular endothelial cell (PMVEC) monolayers. We found that a subpopulation of bone marrow MNCs exposed to 1,000 cGy could be maintained and expanded over 10 days in a PMVEC culture (3.8-fold expansion), whereas liquid suspension culture did not maintain a significant number of hematopoietic cells postirradiation. Colony-forming assays demonstrated that murine MNCs exposed to 1,000 cGy did not give rise to granulocyte/macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-e), or CFU-Mix in 14-day cultures, whereas irradiated MNCs that were subsequently cultured in PMVEC-generated CFU-GM, BFU-e, and CFU-Mix at cloning efficiencies of 1.3%, 0.2%, and 0.2%, respectively. In survival studies, we found that 78% of mice that were irradiated and then transplanted with irradiated/PMVEC-expanded MNCs were alive at day 50, compared with 18% of irradiated control mice (p < 0.05). We also observed that mice transplanted with irradiated/PMVEC-expanded MNCs showed complete hematologic recovery. At 8 weeks post-transplant, we found evidence of Ly 5.1 donor cells in both the bone marrow and the spleen of the transplanted animals, but the levels of engraftment were low (range, 0-5.1%; mean, 1.9%). These results demonstrate that a subpopulation of bone marrow stem cells are capable of surviving the effects of high-dose radiation if these cells are placed in coculture with endothelial cell monolayers.

All-trans retinoic acid facilitates oncoretrovirus-mediated transduction of hematopoietic repopulating stem cells.

A major limiting factor in achieving high levels of gene transfer into hematopoietic stem cells is the ability to retain significant repopulating activity of the stem cells during the ex vivo exposure to oncoretroviral vectors. Recently, we reported that pharmacological levels (1 microM) of all-trans retinoic acid (ATRA) enhanced the maintenance of in vivo repopulating hematopoietic stem cells during liquid suspension culture. Therefore, we investigated the use of ATRA to improve transduction of hematopoietic repopulating cells. Hematopoietic precursors cultured and transduced with a GFP-containing oncoretroviral vector with or without ATRA were transplanted immediately post-transduction (day 3 post-culture initiation) or following extended culture without further transduction (day 7 post-culture initiation). Mice transplanted with 3-day ATRA-treated cells had four-fold more donor cells than the untreated cells. In contrast, there were more GFP-expressing donor cells in recipients of cells cultured without ATRA (31.31 +/- 8.47% no ATRA vs. 16.52 +/- 9.35% ATRA). After 7 days of culture, however, the repopulating ability of the hematopoietic precursors was the same for both treatment groups, but the ATRA-treated cells had significantly more green fluorescence protein (GFP)-expressing donor cells (5.57 +/- O.53% no ATRA vs. 13.67 +/- 2.14% ATRA). Secondary recipients of marrow from recipients of the 3 day cultured cells had similar donor cell levels, but the percentage of GFP-expressing cells within the donor cell population was higher in the recipients of ATRA-treated cells (3.25 +/- 0.70% no ATRA vs. 7.97 +/- 2.71% ATRA). Our data show that the addition of ATRA to cultures of hematopoietic precursors resulted in increased gene transfer into murine hematopoietic repopulating cells. These data suggest that ATRA may be useful in clinical gene therapy protocols using oncoretroviral vectors.

Efficient ex vivo generation of human dendritic cells from mobilized CD34+ peripheral blood progenitors.

We tried to efficiently generate human dendritic cells (DCs) from CD34+ peripheral blood hematopoietic progenitor cells mobilized by high-dose chemotherapy and subsequent administration of granulocyte colony-stimulating factor, using a liquid suspension culture system. Among various combinations, the combination of c-kit ligand, flt-3 ligand, c-mpl ligand (TPO), and interleukin (IL)-4 most potently generated the number of CD1a+CD14- DCs in cultures containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF-alpha). The delayed addition of IL-4 on day 6 of culture gave rise to an additional increase in the yield of CD1a+CD14-DCs that were characterized by the expression of HLA-ABC, HLA-DR, CD80, CD86, and CD83. The majority of the sorted CD1a-CD14+ cells derived from 6-day culture of CD34+ cells gave rise to CD1a+CD14- DCs and CD1a-CD14+ macrophages on day 12 of culture in the presence and absence of IL-4, respectively. These findings suggest that IL-4 promotes the differentiation of CD1a- CD14+ cells derived from mobilized CD34+ peripheral blood hematopoietic progenitors to CD1a+ CD14- DCs. The majority of these DCs expressed CD68 but not the Langerhans-associated granule antigen, a finding that suggests they emerge through the monocyte differentiation pathway. The addition of TPO and IL-4 to cultures did not affect the potential of DCs to stimulate the primary allogeneic T-cell response. These findings demonstrated that the combination of c-kit ligand plus flt-3 ligand plus TPO with GM-CSF plus TNF-alpha, followed by IL-4, is useful for ex vivo generation of human DCs from mobilized CD34+ peripheral blood progenitors.

Enforced expression of the GATA-3 transcription factor affects cell fate decisions in hematopoiesis.

Three GATA family transcription factors are involved in various aspects of hematopoiesis. Their lineage-restricted expression correlates well with their function in selective lineage commitment and differentiation. We focused on the role of GATA-3 to determine whether an intrinsic variation among different GATA proteins, in addition to the distinct expression pattern, determines lineage specification. Using a retroviral vector, we introduced the GATA-3 gene into primary murine hematopoietic stem cells (HSC) and examined their development in in vitro suspension culture and colony-forming assays as well as in vivo competitive repopulation studies. Although GATA-3 expression normally is restricted to lymphoid precursor and committed T cells, overexpression of GATA-3 in HSC results in cessation of cell expansion followed by selective induction of megakaryocytic and erythroid differentiation and inhibition of myeloid and lymphoid precursor development in liquid suspension culture and in vitro colony-forming assays. Competitive repopulation studies show that transplanted GATA-3-expressing HSC/progenitor cells give one wave of erythrocyte development but fail to expand in the bone marrow or to reconstitute other lineages. The selective megakaryocytic/erythroid differentiation in HSC with enforced GATA-3 expression suggests a functional redundancy among GATA proteins and indicates that the specific lineage fate determination by individual GATA proteins is largely regulated at the level of expression in a lineage and developmental-stage restricted fashion, whereas the identity of the GATA factor may not be as important.

Direct seeding of grapevine somatic embryos and regeneration of plants

Somatic embryos of grapevine (Vitis vinifera L.) ‘Chardonnay’ were produced from liquid suspension cultures. Mature somatic embryos were blot dried briefly in the laminar flow hood and germinated directly in Magenta GA-7 Vessels™ containing one of the following potting media: (1) sand, (2) commercial potting mixture (CPM), or (3) CPM overlaid with sand. Each vessel containing 20 ml of distilled water and the potting medium was sterilized by autoclaving for 30 min and cooled overnight before inoculating the somatic embryos. Five somatic embryos were placed in each vessel under aseptic conditions. The vessels were closed and incubated at 26±2°C, 16 h photoperiod at 75 μmol s−1 m−2 light intensity. Results revealed that CPM overlaid with sand was best for plant development. There was more contamination of somatic embryos on pure CPM. Since direct seeding bypasses at least two subcultures in agar medium, it has implications for use of somatic embryos as ‘synthetic seeds’ for clonal plant production. This study shows that somatic embryos of grapevine can be handled with some of the convenience of seeds, emphasizing the feasibility for further automating in vitro plant production, which might be especially useful for new varieties where propagation material is limited.

Stromal cell–derived factor 1α increases polyploidization of megakaryocytes generated by human hematopoietic progenitor cells

AbstractThe alpha chemokine receptor CXCR4 has been shown to be expressed on human hematopoietic progenitor cells and during the megakaryocytic differentiation pathway. Stromal cell–derived factor 1 (SDF-1) is the ligand for CXCR4. In this study, the role of SDF-1α in megakaryocytopoiesis was investigated. CD34+ progenitors purified from peripheral blood were grown in serum-free liquid suspension culture supplemented with thrombopoietin to obtain a virtually pure megakaryocytic progeny. In this condition, the addition of SDF-1α gives rise to megakaryocytes (MKs) showing an increased DNA content and a rise of lobated nuclei, as compared with untreated cells: at day 5, approximately 20% of the cells already showed the presence of more than one nuclear lobe versus fewer than 5% in the control cells; at day 12, approximately 85% of the cells were of large size and markedly polyploid, whereas approximately 60% of the control cells were polyploid, showed fewer lobes, and were a smaller size. This effect was dose-dependent and did not affect the megakaryocytic proliferation. Experiments with the mitogen-activated protein kinase (MAPK) inhibitor PD98059 suggested a role for MAPK pathway on SDF-1α–induced endomitosis. Furthermore, SDF-1α induced a significant increase in the number of proplatelet-bearing MKs and promoted the migration of megakaryocytic cells. Treatment with SDF-1α caused reduction in CXCR4 abundance on the plasma membrane, seemingly owing to receptor internalization. Furthermore, the presence of SDF-1α did not affect the expression of megakaryocytic markers, indicating that differentiation and polyploidization are independently regulated events.

All trans retinoic acid abrogates spontaneous monocytic growth in juvenile chronic myelomonocytic leukaemia.

All trans retinoic acid, the active metabolite of vitamin A, exerts profound effects on cell differentiation. On normal myeloid progenitors, retinoids switch the differentiation program of granulo-macrophagic progenitors towards the granulocytic lineage and consequently reduce CFU-M colony formation. Bone marrow and peripheral blood mononuclear cells from children with Juvenile Chronic Myelomonocytic Leukaemia show typical spontaneous monocytic growth. We questioned whether in this disease, retinoids could switch myelomonocytic growth and inhibit the abnormal CFU-M colony proliferation. Ten JCML samples were studied in the presence of ATRA in methyl cellulose colony assay, before (CFU-C) or after (pre-CFU) liquid suspension culture. In vitro characteristics of JCML such as spontaneous monocytic growth in the absence of growth factor was noted in all patients. In the presence of leucocyte-conditioned medium, nine samples showed only CFU-M growth and one sample CFU-GM growth. Incubation with ATRA inhibited CFU-M colony formation in nine cases. Enhancement of granulocytic differentiation (CFU-G) was noted in nine cases. ATRA also inhibited CD34+ JCML monocytic growth and GM-CSF hypersensitivity. These data suggest that, in JCML progenitors, retinoid pathways are functional and inhibition of immature monocytic progenitors cells may be achieved with retinoids, without impeding granulocytic cell growth.

Mechanisms of differential transferrin receptor expression in normal hematopoiesis.

We have investigated the expression of transferrin receptor (TfR) iron regulatory protein-1 (IRP-1) and iron regulatory protein-2 (IRP-2) in liquid suspension culture of purified hematopoietic progenitor cells (HPCs) induced by a growth factor stimulus to proliferation and unilineage differentiation/maturation through the erythroid, granulocytic, monocytic and megakaryocytic lineages. In initial HPC differentiation, TfR expression is induced in both erythroid and granulopoietic cultures. In late HPC differentiation (i.e. starting from day 5 of culture) and then differentiated precursor maturation, the TfR gene is highly expressed in the erythroid lineage, whereas it is sharply downmodulated in the granulopoietic, monocytopoietic and megakaryocytic series. The elevated TfR expression in erythroid cells is: (a) mediated through a high rate of TfR gene transcription; (b) modulated by intracellular iron levels; (c) mediated by TfR mRNA stabilization through the iron regulatory protein (IRP), in that IRP-1 activity is high in erythroid lineage as compared to the levels observed in other hemopoietic lineages; and (d) dependent on exogenous erythropoietin (Epo) (this is indicated by the marked TfR and IRP-1/IRP-2 downmodulation after Epo starvation). Interestingly, analysis of IRP-1 and IRP-2 expression during hemopoietic differentiation showed that: (a) IRP-1 expression was maintained during all steps of erythroid differentiation, while it was lost in the other hemopoietic lineages; (b) IRP-2 expression was observed during all stages of hemopoietic differentiation in all four lineages. However, IRP-1 and IRP-2 expression and activity are induced when monocytes, which express only low levels of IRP-1 and IRP-2, are induced to maturation to macrophages. These studies indicate that: (a) in normal erythropoiesis, the hyperexpression of TfR, starting from early erythroid HPC differentiation, is Epo-dependent and mediated via transcriptional and post-transcriptional mechanisms; (b) in the granulopoietic, monocytopoietic and megakaryocytic pathways, the TfR is first induced and then downmodulated (the latter phenomenon is mediated via transcriptional suppression of the TfR gene and IRP inactivation).

Mechanisms of differential transferrin receptor expression in normal hematopoiesis

We have investigated the expression of transferrin receptor (TfR) iron regulatory protein-1 (IRP-1) and iron regulatory protein-2 (IRP-2) in liquid suspension culture of purified hematopoietic progenitor cells (HPCs) induced by a growth factor stimulus to proliferation and unilineage differentiation/maturation through the erythroid, granulocytic, monocytic and megakaryocytic lineages. In initial HPC differentiation, TfR expression is induced in both erythroid and granulopoietic cultures. In late HPC differentiation (i.e. starting from day 5 of culture) and then differentiated precursor maturation, the TfR gene is highly expressed in the erythroid lineage, whereas it is sharply downmodulated in the granulopoietic, monocytopoietic and megakaryocytic series. The elevated TfR expression in erythroid cells is: (a) mediated through a high rate of TfR gene transcription; (b) modulated by intracellular iron levels; (c) mediated by TfR mRNA stabilization through the iron regulatory protein (IRP), in that IRP-1 activity is high in erythroid lineage as compared to the levels observed in other hemopoietic lineages; and (d) dependent on exogenous erythropoietin (Epo) (this is indicated by the marked TfR and IRP-1/IRP-2 downmodulation after Epo starvation). Interestingly, analysis of IRP-1 and IRP-2 expression during hemopoietic differentiation showed that: (a) IRP-1 expression was maintained during all steps of erythroid differentiation, while it was lost in the other hemopoietic lineages; (b) IRP-2 expression was observed during all stages of hemopoietic differentiation in all four lineages. However, IRP-1 and IRP-2 expression and activity are induced when monocytes, which express only low levels of IRP-1 and IRP-2, are induced to maturation to macrophages. These studies indicate that: (a) in normal erythropoiesis, the hyperexpression of TfR, starting from early erythroid HPC differentiation, is Epo-dependent and mediated via transcriptional and post-transcriptional mechanisms; (b) in the granulopoietic, monocytopoietic and megakaryocytic pathways, the TfR is first induced and then downmodulated (the latter phenomenon is mediated via transcriptional suppression of the TfR gene and IRP inactivation).

Signal through gp130 Activated by Soluble Interleukin (IL)‐6 Receptor (R) and IL‐6 or IL‐6R/IL‐6 Fusion Protein Enhances Ex Vivo Expansion of Human Peripheral Blood‐Derived Hematopoietic Progenitors

This study was designed to investigate the effects of a combination of soluble interleukin (sIL)-6 receptor (R) and IL-6 on the ex vivo expansion of human peripheral blood (PB)-derived hematopoietic progenitor cells in a short-term serum-free liquid suspension culture system, using PB-derived CD34(+)IL-6R(+/-) cells as a target. In combination with stem cell factor (SCF), IL-3, and sIL-6R/IL-6, the expansion efficiency (EE) for granulocyte/macrophage colony-forming unit (CFU-GM) reached a peak level on day 10 of incubation. On the other hand, the EE for erythroid burst (BFU-E) and mixed colony-forming unit (CFU-Mix) reached a peak level on day 7 of incubation. Among the cytokine combinations tested, SCF + IL-3 + sIL-6R/IL-6 + flt3 ligand (FL) most effectively expanded CFU-GM and CFU-Mix. The maximum EEs for CFU-GM and CFU-Mix were 208-fold and 42-fold, respectively. While the EE for BFU-E was 70-90-fold in the presence of SCF + IL-3 + sIL-6R/IL-6, FL significantly augmented the EE for CFU-GM and CFU-Mix. In contrast, thrombopoietin (TPO) significantly augmented the EE for CFU-Mix. Interestingly, in combination with IL-3 and SCF, newly generated IL-6R/IL-6 fusion protein (FP) expanded PB-derived BFU-E and CFU-Mix twice more effectively than a combination of sIL-6R and IL-6. These results demonstrated that human PB-derived committed progenitors were effectively expanded in vitro using sIL-6R/IL-6 or FP, in combination with IL-3, SCF and/or FL or TPO, and that FP may transduce a stronger intracellular signal than a combination of sIL-6R and IL-6.

Microbial degradation of phenanthrene by addition of a sophorolipid mixture

The influence of sophorolipids on microbial degradation of poorly soluble phenanthrene in liquid and soil suspension culture was evaluated in the work presented. Experiments were carried out in two parts. In the first part, important basic physico-chemical characteristics of the biosurfactant and the pollutant used were determined. The critical micelle concentration (CMC) and the solubilization ratio of the biosurfactant were found to be in a good range compared with synthetic surfactants. Also, a reduction to 71% of the detectable amount of phenanthrene was measured within 4 d in soil suspension without any biotic influence. In the second part, culture experiments were done with Sphingomonas yanoikuyae, the bacterium used throughout the work presented here with the aim to assess the toxicity of the sophorolipids on these bacteria and the effect of the surfactant on biodegradation. In exponential growth tests, no toxicity up to 1 g l −1 sophorolipids could be detected, whereas in an agar plate test, slight growth hindrance was measured at a lower concentration of 250 mg l −1. The above mentioned data were important for planning further experiments. In the following cultivations with liquid and soil suspension media, enhancements of the biodegradation with surfactant addition were measurable. Fluorescence measurements showed that this effect was not due to an increasing biomass, but to an augmentation of bioavailability of the phenanthrene through increasing the apparent dissolved pollutant. Surfactant addition had the consequence of decreasing the residual detectable pollutant concentration (after 36 h 0.5 compared with 2.3 mg l −1 soil suspension) and increasing the maximal degradation rate (127 instead of 80 mg l −1 soil suspension × 10 h). Therefore, the two main problems of biological soil remediation techniques, longer process time and residual pollutants, may be solved by the use of surfactants.

Immunophenotypic and functional characterization of CD33(+)CD34(+) cells in human cord blood of preterm neonates.

To characterize CD33(+)CD34(+) cells, a major population in human cord blood (CB) CD34(+) cells of preterm neonates. The proportion of CD33(+) cells was analyzed on CB CD34(+) cells from preterm and full-term neonates. CD33(+)CD34(+) cells were purified by cell sorting and analyzed on their clonogenic activity, proliferative activity in short-time liquid suspension culture, and GATA-2 mRNA expression by RT-PCR and Southern blot. The absolute numbers and proportion of CD34(+) cells in mononuclear cells inversely correlated with gestational age. CD33 was expressed on a majority of CB CD34(+) cells of preterm neonates but on only a minor population of them in full-term neonates. In addition, CD33 was dominantly expressed on CD38(-)CD34(+) cells or CD117(low)CD34(+) cells in CB of preterm neonates. CD33(+)CD34(+) cells of preterm cord blood had high proliferative and reproducible potentials compared with CD33(-)CD34(+) cells. CD33(+)CD34(+) cells as well as CD33(-)CD34(+) cells from preterm CB highly expressed GATA-2, in contrast to those from BM. These results suggest that CD33(+)CD34(+) cells, which are a major population in CB CD34(+) cells of preterm neonates, do not simply represent relatively mature myeloid lineage hematopoietic progenitor cells as those in adult BM CD34(+) cells, and may contain hematopoietic stem cells or primitive progenitor cells as in fetal liver.

Functional characterization of the two alternative promoters of human p45 NF-E2 gene.

The transcription factor NF-E2, a heterodimeric protein complex composed of p45 and small Maf family proteins, is considered crucial for the proper differentiation of erythrocytes and megakaryocytes in vivo. We report the results of studies aimed at understanding the regulatory mechanisms controlling p45 gene expression in erythroid cells. Human p45 mRNAs have two alternative isoforms, aNF-E2 and fNF-E2, and these isoforms are transcribed from the alternative promoters. We investigated lineage-specific expression of both isomers in human erythroid and megakaryocytic cells by reverse transcriptase polymerase chain reaction or Northern blot analysis. For functional characterization of both promoters, plasmids in which reporter genes were placed under the control of a series of truncated or mutated promoter fragments were transfected to human hematopoietic cell lines. When CD34(+) cells isolated from human cord blood were induced to unilineage erythroid or megakaryocytic differentiation in liquid suspension culture, both transcripts, although barely detected at day 0, were induced in both erythroid and megakaryocytic cultures. fNF-E2 mRNA was found to be more abundant in erythroid cells than megakaryocytic cells at day 7 of culture. Although both isomers were expressed in human erythroid-megakaryocytic cell lines, megakaryocytic maturation with loss of erythroid phenotype induced by phorbol 12-myristate 13-acetate (PMA) resulted in exclusive downregulation of fNF-E2, suggesting that fNF-E2 promoter is more erythroid specific. Functional analysis of fNF-E2 promoter showed that the promoter is active only in erythroid-megakaryocytic cells and that the double GATA site in the proximal region is necessary for its efficient activity. These results suggest that GATA proteins, which govern the differentiation of erythroid lineage cells, are required for full promoter activity of the p45 gene.

Functional characterization of the two alternative promoters of human p45 NF-E2 Gene

The transcription factor NF-E2, a heterodimeric protein complex composed of p45 and small Maf family proteins, is considered crucial for the proper differentiation of erythrocytes and megakaryocytes in vivo. Expression of the p45 subunit is restricted to selected blood lineages, including erythroid and megakaryocytic lineage cells. Here we report results of studies aimed to understand the regulatory mechanisms controlling p45 gene expression in erythroid cells. Human p45 mRNAs have the two alternative isoforms, aNF-E2 and fNF-E2, and these isoforms are transcribed from the alternative promoters. We investigated lineage-specific expression of both NF-E2 isomers by RT-PCR analysis. To examine the expression of both NF-E2 isomers in primary erythroid and megakaryocytic cells, CD34+ cells isolated from human cord blood were induced to unilineage erythroid or megakaryocytic differentiation in liquid suspension culture. fNF-E2 mRNA was found to be more abundant in erythroid cells than megakaryocytic cells, whereas aNF-E2 was predominantly expressed in megakaryocytic cells. Although both isomers were abundantly expressed in human erythroid-megakaryocytic cell lines, megakaryocytic maturation with loss of erythroid phenotype induced by phorbol 12-myristate 13-acetate (PMA) resulted in exclusive down regulation of fNF-E2. A functional analysis of fNF-E2 promoter showed that the promoter is active only in erythroid-megakaryocytic cells and that the double GATA site in the proximal region is necessary for its efficient activity. These results suggest that NF-E2 resides downstream of the GATA proteins in the transcription factor hierarchy that governs the differentiation of erythroid lineage cells.

All-trans retinoic acid enhances the long-term repopulating activity of cultured hematopoietic stem cells

The retinoic acid receptor (RAR) agonist, all-trans retinoic acid (ATRA), is a potent inducer of terminal differentiation of malignant promyelocytes, but its effects on more primitive hematopoietic progenitors and stem cells are less clear. We previously reported that pharmacologic levels (1 micromol) of ATRA enhanced the generation of colony-forming cell (CFC) and colony-forming unit-spleen (CFU-S) in liquid suspension cultures of lin- c-kit+ Sca-1+ murine hematopoietic precursors. In this study, we further investigated the effects of ATRA as well as an RAR antagonist, AGN 193109, on the generation of transplantable cells, including pre-CFU-S, short-term repopulating stem cells (STRCs), and long-term repopulating stem cells (LTRCs). ATRA enhanced the ex vivo maintenance and production of competitive repopulating STRCs and LTRCs from lin- c-kit+ Sca-1+ cells cultured in liquid suspension for 14 days. In addition, ATRA prevented the differentiation of these primitive stem cells into more mature pre-CFU-S during the 14 days of culture. In marked contrast, lin- c-kit+ Sca-1+ cells cultured with AGN 193109 for 7 days had virtually no short- or long-term repopulating ability, but displayed an approximately 6-fold increase in the pre-CFU-S population. The data suggest that the RAR agonist ATRA enhances the maintenance and self-renewal of short- and long-term repopulating stem cells. In contrast, the RAR antagonist AGN 193109 abrogates reconstituting ability, most likely by promoting the differentiation of the primitive stem cells. These results imply an important and unexpected role of retinoids in regulating hematopoietic stem cell differentiation. (Blood. 2000;95:470-477)

Combination of Granulocyte Colony-Stimulating Factor and Low-Dose Cytosine Arabinoside Further Enhances Myeloid Differentiation in Leukemia Cells in Vitro

We examined the differentiation-inducing effect on freshly isolated myeloid leukemia cells in liquid suspension culture by combined treatment with granulocyte colony-stimulating factor (G-CSF) plus low-dose cytosine arabinoside (Ara-C; 5-10 ng/ml) in 25 patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) in leukemic transformation. Culture with G-CSF alone showed leukemic cell growth stimulation in 15 out of the 25 cases (60%) and induction of cell differentiation in 19 out of the 25 cases (76%), respectively. In 23 cases (92%), either growth stimulation and/or differentiation induction of leukemia cells was observed in response to G-CSF. This suggests that most myeloid leukemia cells are able to respond to G-CSF stimulation. In addition, treatment of cells with low-dose Ara-C alone resulted in the enhancement of myeloid specific antigens expression in 16 cases (64%). Treatment of leukemia cells with higher concentrations of Ara-C (over 50 ng/ml) alone resulted in cytocidal effects but not in the induction of differentiation. Furthermore, 15 cases (60%) showed pronounced myeloid differentiation of leukemia cells after combined exposure to G-CSF plus low-dose Ara-C as compared with cells treated with either G-CSF or Ara-C alone. The enhanced effect of differentiation induction by combining G-CSF plus low-dose Ara-C was also observed in a murine myeloid leukemia cell line WEHI-3B in vitro. These data suggest that treatment with G-CSF plus low-dose Ara-C is capable of inducing differentiation of leukemic cells in vitro, and also appears to be useful for the differentiation-based therapy of patients with AML and MDS.

Expression of transcription factors during megakaryocytic differentiation of CD34+ cells from human cord blood induced by thrombopoietin.

Although normal megakaryocytic development has been shown to require the presence of functional GATA-1 and NF-E2 transcription factors in vivo, the roles of other members of the GATA binding factors and NF-E2 family during megakaryocytic differentiation are unclear. the present study, the expression of GATA family members, GATA-1 and GATA-2, a GATA-binding factor, EVI-1, the large subunit of NF-E2 factor, p45 and the related factors, Nrf1, Nrf2, Nrf3, BACH1, BACH2, and the small subunit of NF-E2, MAFK and MAFG has been examined in human megakaryocytic and erythroid cells by reverse transcriptase-polymerase chain reaction. CD34+ cells isolated from human cord blood were induced to unilineage megakaryocytic or erythroid differentiation in liquid suspension culture in the presense of thrombopoietin or erythropoietin, respectively. Each lineage was identified by monoclonal antibody against GPIIb/IIIa or glycophorin A. In megakaryocytic culture, p45, Nrf1, Nrf2, BACH1, MAFK and MAFG mRNAs were induced similarly to erythroid culture. Nrf3 mRNA was barely detected in both cultures. BACH2 was induced only in megakaryocytic culture, although the level of expression was low. Furthermore, the profiles of transcription factors involved in hematopoiesis, EVI-1 and Ets-1 mRNAs were induced only in megakaryocytic culture. Megakaryocytic and erythroid differentiation pathways are closely related to each other, and these two lineage cells share a number of lineage-specific transcription factors. However, the results showed that the profile of the expression of these transcription factors in megakaryocytic cells is distinct from that of erythroid lineage. The dynamic changes in the levels of different transcription factors that occur during primary megakaryocytic differentiation suggest that the levels of these factors may influence the progression to specific hematopoietic pathways.

Rapid Differentiation of a Rare Subset of Adult Human Lin−CD34−CD38− Cells Stimulated by Multiple Growth Factors In Vitro

Recently, several reports of lineage-negative (lin−) CD34− cells with in vivo hematopoietic activity have focused interest on the properties and growth factor response characteristics of these cells. We have now identified a combination of 5 growth factors that are necessary and sufficient to stimulate a marked mitogenic and differentiation response by a subset of human lin−CD34−CD38− cells present in normal adult human marrow and granulocyte colony-stimulating factor (G-CSF)–mobilized blood. Less than 0.1% of the cells in highly purified (including doubly sorted) lin−CD34−CD38− cells from these 2 sources formed colonies directly in semisolid medium or generated such cells after 6 weeks in long-term culture. Nevertheless, approximately 1% of the same lin−CD34−CD38− cells were able to proliferate rapidly in serum-free liquid suspension cultures containing human flt-3 ligand, Steel factor, thrombopoietin, interleukin-3 (IL-3), and hyper–IL-6 to produce a net 28- ± 8-fold increase in total cells within 10 days. Of the cells present in these 10-day cultures, 5% ± 2% were CD34+ and 2.5% ± 0.9% were erythroid, granulopoietic, megakaryocytopoietic, or multilineage colony-forming cells (CFC) (13 ± 7 CFC per lin−CD34−CD38− pre-CFC). In contrast to lin−CD34+CD38−cells, this response of lin−CD34−CD38− cells required exposure to all of the 5 growth factors used. Up to 1.7 × 105 lin−CD34− adult marrow cells failed to engraft sublethally irradiated NOD/SCID-β2M−/− mice. These studies demonstrate unique properties of a rare subset of lin−CD34−CD38− cells present in both adult human marrow and mobilized blood samples that allow their rapid proliferation and differentiation in vitro within an overall period of 3 to 4 weeks. The rapidity of this response challenges current concepts about the normal duration and coordinated control of these processes in adults.

Rapid Differentiation of a Rare Subset of Adult Human Lin−CD34−CD38− Cells Stimulated by Multiple Growth Factors In Vitro

AbstractRecently, several reports of lineage-negative (lin−) CD34− cells with in vivo hematopoietic activity have focused interest on the properties and growth factor response characteristics of these cells. We have now identified a combination of 5 growth factors that are necessary and sufficient to stimulate a marked mitogenic and differentiation response by a subset of human lin−CD34−CD38− cells present in normal adult human marrow and granulocyte colony-stimulating factor (G-CSF)–mobilized blood. Less than 0.1% of the cells in highly purified (including doubly sorted) lin−CD34−CD38− cells from these 2 sources formed colonies directly in semisolid medium or generated such cells after 6 weeks in long-term culture. Nevertheless, approximately 1% of the same lin−CD34−CD38− cells were able to proliferate rapidly in serum-free liquid suspension cultures containing human flt-3 ligand, Steel factor, thrombopoietin, interleukin-3 (IL-3), and hyper–IL-6 to produce a net 28- ± 8-fold increase in total cells within 10 days. Of the cells present in these 10-day cultures, 5% ± 2% were CD34+ and 2.5% ± 0.9% were erythroid, granulopoietic, megakaryocytopoietic, or multilineage colony-forming cells (CFC) (13 ± 7 CFC per lin−CD34−CD38− pre-CFC). In contrast to lin−CD34+CD38−cells, this response of lin−CD34−CD38− cells required exposure to all of the 5 growth factors used. Up to 1.7 × 105 lin−CD34− adult marrow cells failed to engraft sublethally irradiated NOD/SCID-β2M−/− mice. These studies demonstrate unique properties of a rare subset of lin−CD34−CD38− cells present in both adult human marrow and mobilized blood samples that allow their rapid proliferation and differentiation in vitro within an overall period of 3 to 4 weeks. The rapidity of this response challenges current concepts about the normal duration and coordinated control of these processes in adults.

Lineage-Specific Expression of Human Immunodeficiency Virus (HIV) Receptor/Coreceptors in Differentiating Hematopoietic Precursors: Correlation With Susceptibility to T- and M-Tropic HIV and Chemokine-Mediated HIV Resistance

AbstractHuman immunodeficiency virus (HIV) entry is mediated not only by the CD4 receptor, but also by interaction with closely related molecules that act as membrane coreceptors. We have analyzed mRNA expression and/or cell membrane exposition of the coreceptors most widely used by diverse HIV-1 strains (CXCR4, CCR5, and CCR3) on purified hematopoietic progenitor cells (HPCs) induced in liquid suspension culture to unilineage differentiation/maturation through the erythroid (E), granulocytic (G), megakaryocytic (Mk), and monocytic (Mo) lineages. Reverse transcriptase-polymerase chain reaction (RT-PCR) and cytofluorimetric analysis showed the presence of both CXCR4 and CCR5 in quiescent HPCs, but failed to detect CCR3-specific transcripts. Chemokine expression in HPC progenies showed that CXCR4 receptor is detected on the majority of MKs from early to late stages of maturation, whereas it is moderately decreased in the Mo lineage. In the G pathway, two distinct cell populations, CXCR4+ and CXCR4−, were observed: morphological analysis of the sorted populations showed that the CXCR4+ cells were largely eosinophils and the CXCR4− were granulocytes of the neutrophilic series. Furthermore, in the E pathway, CXCR4 was almost completely absent. CCR5 expression is restricted to Mo cultures, ie, ≈30% to 80% cells throughout all monocytopoietic differentiation/maturation stages. Finally, CCR3 mRNA is always absent in all the unilineage cultures. Evaluation of CD4 expression by flow cytometry on both quiescent HPCs and differentiating unilineage precursors showed that the CD4 receptor is present on ≈15% of the starting CD34+ HPC population, highly expressed in the Mo lineage up to 80% at terminal maturation, present on 20% to 30% of maturing Mks, and not detectable in either the E or G lineage. Expression of CD4 receptor together with CXCR4 and/or CCR5 coreceptor in the four lineages correlates with hematopoietic precursor susceptibility to T-lymphotropic and macrophage (M)-tropic HIV strains infection: (1) CD4− G and E cells were resistant to both M-tropic and T-lymphotropic strains; (2) HPC-derived Mks were susceptible to T-tropic, but resistant to M-tropic, infection; (3) Mo differentiating cells efficiently replicate both HIV strains. Furthermore, we showed that the CXCR4 and CCR5 ligands (stromal-derived factor 1 and macrophage-inflammatory protein-1α [MIP-1α], MIP-1β and RANTES, respectively) inhibit HIV replication in both maturing Mo and Mk cells. Taken together, our data show a lineage-specific modulation of chemokine receptor/coreceptor during hematopoietic cell differentiation and extend previous observations on the relationship between the expression of HIV receptor/coreceptors, susceptibility, and chemokine-mediated resistance to HIV infection.